Coated edible plant-derived microvesicle compositions and methods for using the same

ABSTRACT

Compositions are provided that comprise a microvesicle derived from an edible plant. The microvesicles are coated with a plasma membrane derived from a targeting cell and can further be utilized to encapsulate a therapeutic agent. Methods for treating an inflammatory disorder and/or a cancer are further provided and include the step of administering to a subject an effective amount of a composition that includes a microvesicle coated with a plasma membrane derived from a targeting cell.

RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application Ser.No. 61/978,434, filed Apr. 11, 2014, the entire disclosure of which isincorporated herein by this reference.

GOVERNMENT INTEREST

This invention was made with government support under grant numberUH2TR000875 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

TECHNICAL FIELD

The presently-disclosed subject matter relates to coated edibleplant-derived microvesicle compositions and methods of using the samefor the diagnosis and treatment of disease. In particular, thepresently-disclosed subject matter relates to compositions that includeedible plant-derived microvesicles having a targeting cell-derivedplasma membrane coating and that are useful in the treatment of disease.

BACKGROUND

Inflammation is a hallmark of most diseases including cancer, autoimmunedisease, and infectious disease. The development of target-specificdelivery systems to inflammatory sites is urgently needed. Theattraction of leukocytes, including T cells, to sites of inflammationand infection is an essential component of the host response to disease,including autoimmune and chronic inflammatory diseases as well asinfections disease and cancer. Recruitment of circulating T cells tosites of pathogen entry or inflammation involves at least two separatemigration processes, termed extravasation and chemotaxis. Adhesion tothe luminal side of blood vessels, transendothelial migration, andsubsequent chemotaxis of leukocytes are highly complex processes.Chemokines and their receptors play a coordinating role in both thehomeostatic circulation of T cells, as well as their movement to sitesof inflammation or injury. Once T cells are within inflammatory tissue,their response can be affected by the many inflammatory chemokines thatare overexpressed and have broad target cell selectivity. The fact thatthere is a redundancy within the chemokine network with respect toligand-receptor binding and that an array of chemokines areoverexpressed by a variety of cells in inflammatory tissues makes theuse of chemokines a potential component for the development oftherapeutic targeting.

For a therapeutic agent to exert its desired effect it needs to (1)reach the desired site and (2) be in physical contact with its target.The development of target-specific delivery systems has not yet beenbroadly successful. Despite many potential advantages for usingnanoparticles and liposomes, hurdles to their use include cytotoxicity,induction of chronic inflammation, host immune responses, difficultiesof large scale production at affordable prices, and potential biohazardsto the environment. Unlike the situation with artificially synthesizednanoparticles, naturally released nano-sized exosomes derived from manydifferent types of mammalian cells play an important role inintercellular communication. Nano-sized exosomes released from mammaliancells have been utilized for encapsulating drugs and siRNA to treatdiseases in mouse disease models without side-effects. Although thisapproach is promising, production of large quantities of mammalian cellnanoparticles and evaluation of their potential biohazards has beenchallenging. Exosome-like nanoparticles from the tissue of edible plantsincluding grapefruit, grapes, and tomatoes were recently identified, andproduced in large quantities. See, e.g., International PatentApplication Publication No. WO 2013/070324, which is incorporated hereinby this reference. As with mammalian exosomes, it was demonstrated thatexosome-like nanopartides from grapes naturally encapsulate small RNAs,proteins, and lipids. Using both in vitro cell culture models as well asmouse models, it was shown that grapefruit GNVs are highly efficient fordelivering a variety of therapeutic agents including drugs, DNAexpression vectors, siRNA and antibodies. Despite the promise of thosenanovectors, however, efficient and effective targeting of thenanovectors to inflamed and/or diseased tissue remains an issue.

SUMMARY

The presently-disclosed subject matter meets some or all of theabove-identified needs, as will become evident to those of ordinaryskill in the art after a study of information provided in this document.

This Summary describes several embodiments of the presently-disclosedsubject matter, and in many cases lists variations and permutations ofthese embodiments. This Summary is merely exemplary of the numerous andvaried embodiments. Mention of one or more representative features of agiven embodiment is likewise exemplary. Such an embodiment can typicallyexist with or without the feature(s) mentioned; likewise, those featurescan be applied to other embodiments of the presently-disclosed subjectmatter, whether listed in this Summary or not. To avoid excessiverepetition, this Summary does not list or suggest all possiblecombinations of such features.

The presently-disclosed subject matter includes coated edibleplant-derived microvesicle compositions and methods of using the samefor the diagnosis and treatment of disease. In particular, thepresently-disclosed subject matter includes compositions that includeedible plant-derived microvesicles having a targeting cell-derivedplasma membrane coating and that are useful in the treatment of disease.

In some embodiments of the presently-disclosed subject matter, acomposition is provided that comprises a microvesicle derived from anedible plant, where the microvesicle is, in turn, coated with a plasmamembrane derived from a targeting cell. In some embodiments, the edibleplant is a fruit or a vegetable, such as, in some embodiments, a grape,a grapefruit, or a tomato. In some embodiments, the targeting cell usedto derive the plasma membrane coating the microvesicle is an activatedleukocyte.

With further respect to the microvesicle compositions, in someembodiments, the microvesicle encapsulates a therapeutic agent. In someembodiments, the therapeutic agent is selected from a phytochemicalagent and a chemotherapeutic agent. In some embodiments, thephytochemical agent is selected from curcumin, resveratrol, baicalein,equol, fisetin, and quercetin. In some embodiments, the chemotherapeuticagent is selected from the group consisting of retinoic acid,5-fluorouracil, vincristine, actinomycin D, adriamycin, cisplatin,docetaxel, doxorubicin, and taxol. In other embodiments, the therapeuticagent comprises a nucleic acid molecule, such as, in some embodiments,an siRNA, a micro RNA, or a mammalian expression vector. In someembodiments, pharmaceutical compositions are further provided where themicrovesicle compositions are combined with apharmaceutically-acceptable vehicle, carrier, or excipient.

Further provided, in some embodiments of the presently-disclosed subjectmatter, are methods for treating an inflammatory disorder. In someembodiments, a method for treating an inflammatory disorder is providedthat comprises the step of administering to a subject in need thereof aneffective amount of a microvesicle composition described herein. In someembodiments, the inflammatory disorder is selected from the groupconsisting of sepsis, septic shock, colitis, colon cancer, andarthritis. For administration of the compositions, in some embodiments,the composition is administered orally or intransally. In someembodiments, subsequent to administration, the composition reduces anamount of an inflammatory cytokine in a subject, such as, in certainembodiments, tumor necrosis factor-α, interleukin-1β, interferon γ, andinterleukin-6.

Still further provided, in some embodiments of the presently-disclosedsubject matter, are methods for treating a cancer. In some embodiments,a method for treating a cancer in a subject in a subject is providedthat comprises administering to a subject an effective amount of amicrovesicle composition described herein. In some embodiments of themethods for treating a cancer, the therapeutic agent is selected from aphytochemical agent and a chemotherapeutic agent. In some embodiments,the cancer is selected from a brain cancer, a breast cancer, a lungcancer, and a colon cancer.

Further features and advantages of the present invention will becomeevident to those of ordinary skill in the art after a study of thedescription, figures, and non-limiting examples in this document.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D include schematic diagrams, graphs, and images showing thecharacterization of inflammatory cell plasma membrane-coatedgrapefruit-derived nanovectors GNVs (IGNVs). FIG. 1A is a schematicdiagram showing the preparation process of the IGNVs and drug loaded-GNVmicrovesicles for targeted delivery of therapeutic agents toinflammatory sites. FIG. 1B is a graph showing the size distribution andsurface Zeta potential of free GNVs and EL4 cells plasma membrane coatedGNVs (IGNVs) were measured using a ZetaSizer. FIG. 1C includes imagesshowing free GNVs (left) and IGNVs (right) that were visualized andimaged by scanning electron microscopy. FIG. 1D includes images showingthe co-localization of the EL4 cell derived plasma membranes and GNVcores, where, for assembling IGNVs, the EL4 cell derived plasmamembranes were labeled with PKH67 green dye and GNV cores were labeledwith PKH26 red dye, where 4T1 cells were cultured in the presence ofGNVs (upper panel) or IGNVs (bottom panel) for 12 h, and whererepresentative images of cells were then taken using a confocalmicroscope at a magnification of ×400. FIG. 1E is a graph showing FRETbased measurements of IGNV formation, where DiO labeled GNVs and DPAlabeled membrane vesicles (n=3) were mixed, where the mixture wassubsequently extruded 20 times through a 200 nm polycarbonate porousmembrane using an Avanti mini extruder or the mixture without furtherextrusion was used as a control, and where the extruded products and themixed products were then diluted and the intensity of fluorescence wasmeasured.

FIGS. 2A-2E include images and graphs showing the ability of IGNVs toutilize the activated leukocyte membrane dependent pathways andefficiently target and be delivered to inflammatory sites. FIG. 2Aincludes images and graphs showing the results of a transwell assay fordetecting chemotaxis of EL4 cell plasma membrane coated GNVs, whereHUVEC cells were cultured in the upper chamber and 4TO7 cells werecultured in the lower chamber of a transwell plate, where transmigrationof the PKH26 labeled GNVs or IGNVs were imaged after 24 h and 48 h inculture using a confocal microscope, and where the intensity of thefluorescent signal of media in the lower chamber (n=3) was measured andexpressed as the percent of transwell efficiency of fluorescentintensity of PKH26 labeled GNVs or IGNVs. FIGS. 2B-2E are images andgraphs showing the distribution of DiR dye labeled IGNVs in: LPS inducedskin acute inflammatory mouse model (FIG. 2B); DSS induced colitis mice(FIG. 2C); CT26 tumor model (FIG. 2D); and 4T1 tumor model (FIG. 2E);where live-mouse images (left) were collected 6 h and 24 h after I. V.injection of DiR dye labeled IGNVs, where skin, colon, and tumor tissueswere removed 24 h after the injection and scanned for DiR dye signals,and where a representative image from each group of mice is shown (leftpanels) and followed by graphical figures (right panels) presented asthe mean net intensity (Sum Intensity/Area, n=5) (**p<0.01 and***p<0.001).

FIGS. 3A-3I include images and graphs showing the ability of chemokinemediated pathways to play a causative role in the efficient targeteddelivery of IGNVs to inflammatory sites. FIG. 3A includes images showingchemokine expression in normal skin (Normal), LPS induced acuteinflammatory skin tissues (Skin-LPS), CT26 (CT26 tumor) and 4T1 (4T1tumor) tissues, where the expressions were determined using a ProteomeProfiler from R&D systems, and where each dot represents a chemokinedetected by a capture antibody and printed in duplicate on a membrane.FIG. 3B includes graphs showing the expression of chemokine receptors onIGNVs, where the IGNVs were analyzed by FACS analysis of IGNVs coated on4 μm-diameter aldehyde/sulfate latex beads. FIG. 3C is a graph showingin vitro transmigration of IGNVs, where HUVEC cells (n=3) were culturedin the fibronectin coated-upper chamber as a transmigration barrier,where PKH26 labeled IGNVs (PKH26-IGNVs) were pre-incubated withrecombinant chemokines as listed in FIG. 1C or with the extract from 4T1tumor, where, after washing, pre-incubated PKH26-IGNVs were added to theupper chamber, and cultured in the presence of recombinant chemokines(CXCLI/2/9/10 plus CCL2/5) in the lower chamber, and where, after a 24 hincubation, the intensity of PKH26 fluorescence of media in the lowerchamber (n=3) was measured and expressed as the % of transwellefficiency of PKH26⁺ IGNVs. FIG. 3D includes images and a graph showingIGNVs in LPS-induced acute skin inflammatory mice, where DiR dye labeledIGNVs were pre-incubated overnight at 4° C. with (neutralized) orwithout (not neutralized) 4T1 extract before an I.V. injection, andwhere a representative image at 6 h and 24 h after the injection fromeach group of mice (n=5) is shown (left) and followed by graphicalfigures (right) presented as the mean net intensity (Sum Intensity/Area,n=5). FIG 3E includes images showing immunohistochemical staining ofchemokines (CCL2, CCL5, CXCL9 and CXCL10) expressed in human breastcancer, colon cancer tissues (bottom panels) and paired adjacent tissues(upper panels), where a representative image (n=20 for colon cancer,n=21 for breast cancer) from each sample is shown. FIGS. 3F-3G aregraphs showing the results of experiments where DiR dye labeled IGNVswere pre-incubated at 4° C. overnight with recombinant chemokines aslisted in the figures and then I.V. injected into LPS-induced acute skininflammatory mice (FIG. 3F) or CT26 tumor-bearing mice (FIG. 3G), whereDiR dye signals in skin and tumor tissues were determined 24 h after theinjection. FIG. 3H is a graph showing transmigration of IGNVswith/without LFA-1 neutralization, where PKH26 labeled IGNVs werepre-incubated overnight with anti-LFA-1 antibody at 4° C. and then addedinto the apical chamber, and where the intensity of PKH26 fluorescenceof the media in the lower chamber was measured after 24 and 48 h ofincubation and expressed as the % of transwell efficiency of PKH26⁺IGNVs. FIG. 3I includes an image and a graph showing the results of anexperiment where DiR dye labeled IGNVs were pre-incubated withfunctional anti-LFA-1 antibody at 4° C. overnight, washed, I.V. injectedinto LPS-induced acute skin inflammatory mice, and the DiR signals wasdetected after 24 h injection.

FIGS. 4A-4E include images and graphs showing the targeting to humancolon cancer by coating GNVs with the plasma membrane of LPS stimulatedleukocytes isolated from peripheral blood of healthy human subjects(hIGNVs) or of mice (mIGNVs). FIGS. 4A-4B include graphs showing theprofiles of hIGNV (FIG. 4A) and mIGNV (FIG. 4B) chemokine receptorsbased on FACS analysis, where representative histograms (n=5) show thepercentage of staining of chemokine receptors from the hIGNVs andmIGNVs, and where three different bands from sucrose gradients of plasmamembrane from LPS stimulated leukocytes were used for coating GNVs: topband (LPS-T), middle band (LPS-M), and bottom band (LPS-B). FIG. 4Cincludes images and graphs showing trafficking of DiR dye labeled hIGNVsin human colon cancer SW620-bearing mice, where mice were I.V. injectedwith DiR dye labeled hIGNVs, where live imaging of whole mice wascarried out on day 1 and 5 after the injection, where, at day 5 afterthe injection, tumors were removed and scanned, and where trafficking ofDiR dye labeled-mIGNVs in LPS-induced an acute skin inflammatory mousemodel were measured. FIGS. 4D-4E includes images and a graph showing theresults of an experiment where mice were I.V. injected with DiR dyelabeled mIGNVs without (FIG. 4D) or with (FIG. 4E) CXCR2 knockout, whereskin was removed 72 h (FIG. 4D) or 24 h (FIG. 4E) after the injectionand scanned. A representative image (FIGS. 4C-4D) from each group ofmice is shown and graphical figures are presented as the mean netintensity (Sum Intensity/Area, n=5). *p<0.05, **p<0.01 and ***p<0.001.

FIGS. 5A-5F includes graphs and images showing targeted therapeutic drugdelivery for mouse cancer and colitis therapy. FIG. 5A is a graphshowing stability of circulating IGNVs. FIG. 5B includes images showingin vitro release profile of doxorubicin from IGNV-DOX in PBS buffer withdifferent pH values (5.0, 5.5, 6.0, 6.5 and 7.2, n=5, **p<0.01) FIG. 5Cis a graph showing biodistribution of doxorubicin, in 4T1 tumor-bearingmice, where 4T1 tumor-bearing mice (n=5) were I.V. injected withIGNV-DOX or DOX-NP™, and where the doxorubicin in 4T1 tumor tissues,livers, lungs, spleens, kidneys, hearts and thymus was measured(*p<0.05). FIG. 5D includes images showing biodistribution ofdoxorubicin in CT26 and 4T1 tumor tissues, where free doxorubicin (FreeDOX), GNVs delivered doxorubicin (GNV-DOX) and IGNVs delivereddoxorubicin (IGNV-DOX) were I.V. injected into CT26 (n=5) and 4T1tumor-bearing mice (n=5), where tumor tissues were removed, fixed andsectioned, and where doxorubicin in tumor tissues was observed using aconfocal imaging system. FIGS. 5E-5F includes images and graphs showingthe results of experiments where CT26 and 4T1 cells were injectedsubcutaneously (CT26) or in a mammary fat pad (4T1) of BALB/c mice,where mice were I.V. injected with IGNV-DOX or controls as listed in thefigure every 3 days for 30 days from 7 days after tumor cells wereinjected, where representative images of tumors (FIG. 5E, left panel)from each group (n=5) are shown, tumor volume was measured every 5 days,(FIG. 5E, right panel), and where the survival rate (FIG. 5F) of micewas calculated (*p<0.05, and **p<0.01).

FIGS. 6A-6B include images and graphs showing chemokine receptorsexpressed on PMA stimulated EL4 cells. FIG. 6A includes images showingthe morphology of cultured EL4 cells without (left) or with (right) PMAstimulation, original magnification×10. FIG. 6B includes graphs showingthe percentages of chemokine receptors expressed on EL4 cellswith/without PMA stimulation, where EL4 cells (n=3) were harvested at 24h after PMA stimulation and stained with fluorescence labeled anti-CCR3,CCR4, CCR5, CCR7, CCSR9, CXCR3 and CXCR7 antibodies and the percentageof chemokine receptors were analyzed by FACS.

FIG. 7 is an image showing separation of EL4 plasma membrane viadiscontinuous sucrose gradient centrifugation, where EL4 cellswith/without PMA stimulation were harvested and homogenized, and wherethe homogenized samples were sucrose banded and the images were takenafter the centrifugation.

FIG. 8 includes images showing the uptake of a mixture of GNVs andmembrane derived vesicles, where EL4 cell derived plasma membranevesicles were labeled with PKH67 and GNVs were labeled with PKH26, where4T1 cells were cultured in the presence of simply mixed PKH67-membranevesicles and PKH26-GNVs for 12 h, and where representative images ofcells (n=3) were then taken using a confocal microscope at amagnification of ×400.

FIGS. 9A-9B include graphs showing the stability of IGNVs, where thestability of EL4 cell membrane coated GNVs (n=5) at 22° C. over a 5 dayperiod (FIG. 9A) or at 37° C. over 25 h (FIG. 9B) were analyzed bymeasuring the size distribution and surface zeta potential.

FIG. 10 includes graphs showing the transmigration of GNVs and IGNVs,where HUVEC cells were cultured in the fibronectin-coated (7.5 μg/cm²)upper chamber as a transmigration barrier, where PKH26 dye labeled GNVs(upper panels) or IGNVs (lower panels) were added into the upper chamberand incubated for 24 h and 48 h, and where the PKH26 dye labeledparticles in the medium of the lower chamber were measured, andexpressed as a percentage of transwell efficiency of PKH26⁺ IGNVs.

FIG. 11 includes graphs showing the expression of integrals on EL4cells, where EL4 cells were activated with PMA and the expression ofintegrins (LFA-1 and α4β7) on the cells was detected by staining withPE-anti-LFA-1 and α4β7 antibodies and the percentage ofintegrin-positive cells were analyzed by FACS.

FIG. 12 includes graphs showing LFA-1 on IGNVs, where IGNVs were freshlyprepared and conjugated with latex beads, and where the percentage ofLFA-1 positive IGNVs was quantified by FACS analysis of PE-anti-LFA-1stained IGNVs.

FIGS. 13A-13B include images showing the purification of plasmamembranes from leukocytes, where leukocytes from peripheral blood ofhumans and mice were isolated and stimulated in vitro with (+LPS) (100ng/ml) or PBS as a control (−LPS) for 24 h, and where the homogenizedplasma membranes of cells from human (FIG. 13A) and mouse (FIG. 13B)were purified by sucrose gradient centrifugation (T=top band of sucrosegradient purified membrane, M=middle band of sucrose gradient purifiedmembrane, B=bottom band of sucrose gradient purified membrane).

FIG. 14 includes graphs showing chemokines in the culture medium ofhuman SW620 cells, where human colorectal adenocarcinoma SW620 cellswere cultured in 6-well plates for 48 h and CCL2, CCL5 and CXCL10chemokine release into the culture supernatants was quantitativelyanalyzed with an ELISA.

FIGS. 15A-15F include images and graphs showing the effects of IGNVs oncell proliferation in vitro and potential toxicity in vivo. FIG. 15A isa graph showing the results of an experiment where the proliferation ofIGNV treated-4T1 cells was analyzed using the ATPlite assay 24 h afterexposure to different concentration of IGNVs. FIG. 15B is a graphshowing the results of an experiment where numbers of living 4T1 cellstreated with IGNVs at passage number 1 through 5 were determined bycounting the number of living cells (unstained with trypan blue). FIG.15C-15D include graphs showing the results of an experiment where serumTNF-α, IL-6 and IL-1β (FIG. 15C) and ALT, AST (FIG. 15D) were analyzed24 h after mice were I.V. injected with IGNVs three times or PBS as acontrol (normal control). FIG. 15E includes images of H&E-stainedsections of livers, spleens, kidneys and lungs from IGNV-injected mice,original magnification×20. FIG. 15F includes graphs showing the liver,lung, spleen, kidney, and heart weights of injected animals.

FIGS. 16A-16E include images and graphs showing therapeutic drug-loadedIGNVs. FIG. 16A is an image showing purification of therapeutic drugs(left: doxorubicin, right: curcumin) loaded into IGNVs by discontinuoussucrose density gradient centrifugation, FIGS. 16B-16C are graphsshowing size distribution (FIG. 16B) and Zeta potential (FIG. 16C) ofIGNV-DOX and IGNV-Cur analyzed using a ZetaSizer. FIG. 16B is a graphshowing the loading efficiency of doxorubicin and curcumin detected byUV spectrometry and expressed as %=(Total drugs−free drugs)/Totaldrugs×100%. FIG. 16E includes graphs showing the release profile ofdoxorubicin and curcumin in PBS buffer at pH 7.2 and 37° C. as detectedby UV spectrometry at 497 and 426 nm, respectively.

FIGS. 17A-17D include graphs showing the characterization of DOX-NP™ andcontrol liposomes from Avanti. FIG. 17A is a graph showing the profileof doxorubicin released from DOX-NP™ at 37° C. in PBS buffer atdifferent pH values (5.0, 5.5, 6.0, 6.5 and 7.2). FIG. 17B is a graphshowing the proliferation of 4T1 cells treated with differentconcentrations of control liposomes and analyzed using the ATPIiteassay. FIGS. 17C-17D are graphs showing the results of an experimentwhere mice were I.V. injected with control liposomes daily for 3 days,where 24 h after the last injection of control liposomes, serum TNF-α,IL-6 and IL-1β (FIG. 17C) and the liver enzymes AST and ALT (FIG. 17D)were assessed (n=5 mice per group).

FIGS. 18 includes graphs showing the biodistribution of DOX in 4T1 tumortissues, where DOX (200 μg) was loaded in the GNVs (GNV-DOX), thenGNV-DOX was mixed or extruded with EL4 T cell derived membranes(IGNV-DOX), where, after washing, the GNV-DOX or IGNV-DOX were I.V.injected into 4T1 tumor-bearing mice, and where 24 hours later, micewere sacrificed, tumor tissues were removed and the DOX in tumor tissueswere extracted and detected.

FIGS. 19A-19E include images and graphs showing the therapeutic effectof IGNV-Cur on the inhibition of DSS-induced mouse colitis, whereDSS-induced colitis mice were I.V. injected with PBS, GNVs, IGNVs, freecurcumin, GNV-Cur or IGNV-Cur every 2 days for 10 days, where at day 0after the last treatment, the degree of rectal prolapse and intestinalbleeding was determined (FIG. 19A), body weight was measured daily (FIG.19B), survival rates were analyzed (FIG. 19C), sectioned colon tissueswere H&E stained (n=5) (FIG. 19D), and curcumin in colon tissues wasquantified by HPLC (FIG. 19E).

FIG. 20 includes graphs showing the therapeutic effect of IGNV-Cur onthe inhibition of cytokine induction from colitis tissue, where, day 3after being provided with 2.5% DSS, mice (n=5 mice per group) were I.V.injected with PBS, free GNVs (200 nmol), IGNVs (200 nmol), free Cur (50mg/kg), GNV-Cur (50 mg/kg) or IGNV-Cur (50 mg/kg), every 2 days for 5times, and where 24 h after the last injection, serum TNF-α, IL-6 andIL-1β were determined by ELISA.

FIG. 21 is a graph showing the determination of GNV micellerconcentration (cmc), where different concentrations of total lipids(X-axis) extracted from grapefruit nanoparticles were used forgenerating lipid film, and where, after a bath-sonication, the formationand size distribution of GNV (nm) was analyzed using a ZetaSizer.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

The details of one or more embodiments of the presently-disclosedsubject matter are set forth in this document. Modifications toembodiments described in this document, and other embodiments, will beevident to those of ordinary skill in the art after a study of theinformation provided in this document. The information provided in thisdocument, and particularly the specific details of the describedexemplary embodiments, is provided primarily for clearness ofunderstanding and no unnecessary limitations are to be understoodtherefrom. In case of conflict, the specification of this document,including definitions, will control.

While the terms used herein are believed to be well understood by one ofordinary skill in the art, definitions are set forth herein tofacilitate explanation of the presently-disclosed subject matter.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the presently-disclosed subject matter belongs.Although any methods, devices, and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresently-disclosed subject matter, representative methods, devices, andmaterials are now described.

Following long-standing patent law convention, the terms “a”, “an”, and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “a cell” includes aplurality of such cells, and so forth.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as reaction conditions, and so forth usedin the specification and claims are to be understood as being modifiedin all instances by the term “about”. Accordingly, unless indicated tothe contrary, the numerical parameters set forth in this specificationand claims are approximations that can vary depending upon the desiredproperties sought to be obtained by the presently-disclosed subjectmatter.

As used herein, the term “about,” when referring to a value or to anamount of mass, weight, time, volume, concentration or percentage ismeant to encompass variations of in some embodiments ±20%, in someembodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, insome embodiments ±0.5%, and in some embodiments ±0.1% from the specifiedamount, as such variations are appropriate to perform the disclosedmethod.

As used herein, ranges can be expressed as from “about” one particularvalue, and/or to “about” another particular value. It is also understoodthat there are a number of values disclosed herein, and that each valueis also herein disclosed as “about” that particular value in addition tothe value itself. For example, if the value “10” is disclosed, then“about 10” is also disclosed. It is also understood that each unitbetween two particular units are also disclosed. For example, if 10 and15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

The presently-disclosed subject matter is based, at least in part, onthe discovery that the binding and coating of inflammatory cell-derivedmembranes on grapefruit-derived microvesicles or nanovectors (GNVs) isan effective and efficient strategy to take advantage of the unlimitedavailability of GNVs and to generate personalized delivery vectors thatwould target inflammatory sites in diseases (FIG. 1A). As describedherein below, such compositions have, among others, two advantages,namely; 1) the plasma membrane from activated leukocytes ispreferentially bound on the microvesicles made of fruit nanoparticleslipids; and 2) the resultant microvesicles are safe and can besuccessfully used for targeted delivery of therapeutic agents toinflammatory sites.

Microvesicles are naturally existing nanoparticles that are in the formof small assemblies of lipid particles, are about 50 to 1000 nm in size,and are not only secreted by many types of in vitro cell cultures and invivo cells, but are commonly found in vivo in body fluids, such asblood, urine and malignant ascites. Indeed, microvesicles include, butare not limited to, particles such as exosomes, epididimosomes,argosomes, exosome-like vesicles, microparticles, promininosomes,prostasomes, dexosomes, texosomes, dex, tex, archeosomes, and oncosomes.

As noted above, microvesicles can be formed by a variety of processes,including the release of apoptotic bodies, the budding of microvesiclesdirectly from the cytoplasmic membrane of a cell, and exocytosis frommultivesicular bodies. For example, exosomes are commonly formed bytheir secretion from the endosomal membrane compartments of cells as aconsequence of the fusion of multivesicular bodies with the plasmamembrane. The multivesicular bodies are formed by inward budding fromthe endosomal membrane and subsequent pinching off of small vesiclesinto the luminal space. The internal vesicles present in the MVBs arethen released into the extracellular fluid as so-called exosomes.

As part of the formation and release of microvesicles, unwantedmolecules are eliminated from cells. However, cytosolic and plasmamembrane proteins are also incorporated during these processes into themicrovesicles, resulting in microvesicles having particle sizeproperties, lipid bilayer functional properties, and other uniquefunctional properties that allow the microvesicles to potentiallyfunction as effective nanoparticle carriers of therapeutic agents. Inthis regard, the term “microvesicle” is used interchangeably herein withthe terms “nanoparticle,” “liposome,” “exosome,” “exosome-likeparticle,” “nano-vector” and grammatical variations of each of theforegoing. It has now been discovered though that edible plants, such asfruits, are not only a viable source of large quantities ofmicrovesicles, but that microvesicles derived from edible plants can becoated with plasma membranes derived from targeting cells, such asleukocytes, and used as an effective delivery vehicle for homing orotherwise targeting the microvesicles, including any therapeutic agentsthat may be included within the microvesicles, to the sites ofinflammation and disease.

The presently-disclosed subject matter thus includes edibleplant-derived microvesicle compositions that are coated with plasmamembranes derived from targeting cells. The term “targeting cells,” andgrammatical variations thereof, is used herein to refer to those cellsthat, by virtue of the inclusion of certain targeting moieties (e.g.,receptors or other proteins) in their plasma membranes, preferentiallyhome or are trafficked to certain tissues and organs in the body of asubject. For example, in some embodiments, the targeting cells areactivated inflammatory cells, such as leukocytes, or macrophages orneutrophils that preferentially target or home to sites of inflammation.As another example, in some embodiments, the targeting cells arecirculating tumor cells or metastatic tumor cells that preferentiallyhome to tissues such as brain, lung, liver, and bone tissue (i.e.,frequent sites of metastasis). As yet another example, in someembodiments, the targeting cells are stem cells (e.g., bone marrowderived stem cells) that can be preferentially targeted to and used fortissue regeneration.

In some embodiments, the microvesicle compositions further includetherapeutic agents and are useful in the treatment of various diseases,including inflammatory disorders and cancers. In some embodiments of thepresently-disclosed subject matter, a microvesicle composition isprovided that comprises a microvesicle derived from an edible plant anda plasma membrane coating on the microvesicle, where the plasma membranehas been derived from a targeting cell. In some embodiments, acomposition is provided wherein a therapeutic agent is encapsulated bythe microvesicle derived from the edible plant. In some embodiments, thetherapeutic agent encapsulated by the edible-plant derived microvesicleis selected from a phytochemical agent and a chemotherapeutic agent.

The term “edible plant” is used herein to describe organisms from thekingdom Plantae that are capable of producing their own food, at leastin part, from inorganic matter through photosynthesis, and that are fitfor consumption by a subject, as defined herein below. Such edibleplants include, but are not limited to, vegetables, fruits, nuts, andthe like. In some embodiments of the microvesicle compositions describedherein, the edible plant is a fruit. In some embodiments, the fruit isselected from a grape, a grapefruit, and a tomato.

The phrase “derived from an edible plant,” when used in the context of amicrovesicle derived from an edible plant, refers to a microvesiclethat, by the hand of man, exists apart from its native environment andis therefore not a product of nature. In this regard, in someembodiments, the phrase “derived from an edible plant” can be usedinterchangeably with the phrase “isolated from an edible plant” todescribe a microvesicle of the presently-disclosed subject matter thatis useful for being coated with an inflammatory cell-derived plasmamembrane and/or for encapsulating therapeutic agents.

The phrase “encapsulated by a microvesicle,” or grammatical variationsthereof is used herein to refer to microvesicles whose lipid bilayersurrounds a therapeutic agent. For example, a reference to “microvesiclecurcumin” refers to an microvesicle whose lipid bilayer encapsulates orsurrounds an effective amount of curcumin. In some embodiments, theencapsulation of various therapeutic agents within microvesicles can beachieved by first mixing the one or more of the phytochemical agents orchemotherapeutic agents with isolated microvesicles in a suitablebuffered solution, such as phosphate-buffered saline (PBS). After aperiod of incubation sufficient to allow the therapeutic agent to becomeencapsulated during the incubation period, the microvesicle/therapeuticagent mixture is then subjected to a sucrose gradient (e.g., and 8, 30,45, and 60% sucrose gradient) to separate the free therapeutic agent andfree microvesicles from the therapeutic agents encapsulated within themicrovesicles, and a centrifugation step to isolate the microvesiclesencapsulating the therapeutic agents. After this centrifugation step,the microvesicles including the therapeutic agents are seen as a band inthe sucrose gradient such that they can then be collected, washed, anddissolved in a suitable solution for use as described herein below.

As noted, in some embodiments, the therapeutic agent is a phytochemicalagent. As used herein, the term “phytochemical agent” refers to anon-nutritive plant-derived compound, or an analog thereof. Examples ofphytochemical agents include, but are not limited to compounds such asmonophenols; flavonoids, such as flavonols, flavanones, flavones,flavan-3-ols, anthocyanins, anthocyanidins, isoflavones,dihydroflavonols, chalcones, and coumestans; phenolic acids;hydroxycinnamic acids; lignans; tyrosol esters; stillbenoids;hydrolysable tannins; carotenoids, such as carotenes and xanthophylls;monoterpenes; saponins; lipids, such as phytosterols, tocopherols, andomega-3,6,9 fatty acids; diterpenes; triterpinoids; betalains, such asbetacyanins and betaxanthins; dithiolthiones; thiosulphonates; indoles;and glucosinolates. As another example of a phytochemical agentdisclosed herein, the phytochemical agent can be an analog of aplant-derived compound, such as oltipraz, which is an analog of1,2-dithiol-3-thione, a compound that is found in many cruciferousvegetables.

In some embodiments of the presently-disclosed subject matter, thetherapeutic agent is a phytochemical agent selected from curcumin,resveratrol, baicalein, fisetin, and quercetin. In some embodiments, thephytochemical agent is curcumin. Curcumin is a pleiotropic naturalpolyphenol with anti-inflammatory, anti-neoplastic, anti-oxidant andchemopreventive activity, with these activities having been identifiedat both the protein and molecular levels. Nevertheless, limited progresshas been reported with respect to the therapeutic use of curcumin ascurcumin is insoluble in aqueous solvents and is relatively unstable. Inaddition, curcumin is known to have a low systemic bioavailability afteroral dosing, which further limits its usage and clinical efficacy. Ithas been determined, however, that by encapsulating curcumin in edibleplant derived microvesicles, not only can the solubility of curcumin beincreased, but the encapsulation of the curcumin within themicrovesicles protects the curcumin from degradation and also increasesthe bioavailability of the microvesicle curcumin.

As also noted herein above, in some embodiments of thepresently-disclosed subject matter, the therapeutic agent that isencapsulated within the exosome is a chemotherapeutic agent. Examples ofchemotherapeutic agents that can be used in accordance with thepresently-disclosed subject matter include, but are not limited to,platinum coordination compounds such as cisplatin, carboplatin oroxalyplatin; taxane compounds, such as paclitaxel or docetaxel;topoisomerase I inhibitors such as camptothecin compounds for exampleirinotecan or topotecan; topoisomerase II inhibitors such as anti-tumorpodophyllotoxin derivatives for example etoposide or teniposide;anti-tumor vinca alkaloids for example vinblastine, vincristine orvinorelbine; anti-tumor nucleoside derivatives for example5-fluorouracil, gemcitabine or capecitabine; alkylating agents, such asnitrogen mustard or nitrosourea for example cyclophosphamide,chlorambucil, carmustine or lomustine; anti-tumor anthracyclinederivatives for example daunorubicin, doxorubicin, idarubicin ormitoxantrone; HER2 antibodies for example trastuzumab; estrogen receptorantagonists or selective estrogen receptor modulators for exampletamoxifen, toremifene, droloxifene, faslodex or raloxifene; aromataseinhibitors, such as exemestane, anastrozole, letrazole and vorozole;differentiating agents such as retinoids, vitamin D and retinoic acidmetabolism blocking agents (RAMBA) for example accutane; DNA methyltransferase inhibitors for example azacytidiae; kinase inhibitors forexample flavoperidol, imatinib mesylate or gefitinib;farnesyltransferase inhibitors; HDAC inhibitors; other inhibitors of theubiquitin-proteasome pathway for example VELCADE® (MillenniumPharmaceuticals, Cambridge, Mass.); or YONDELIS® (Johnson & Johnson, NewBrunswick, N.J.). In some embodiments, the chemotherapeutic agent thatis encapsulated by an exosome in accordance with the presently-disclosedsubject matter is selected from retinoic acid, 5-fluorouracil,vincristine, actinomycin D, adriamycin, cisplatin, docetaxel,doxorubicin, and taxol.

In other embodiments of the presently-disclosed subject matter,therapeutic agents included within the microvesicle compositionscomprises nucleic acid molecules selected from a siRNA, a microRNA, andan expression vector, such as a mammalian expression vector. The term“nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single- or double-stranded form. Unlessspecifically limited, the term encompasses nucleic acids containingknown analogues of natural nucleotides that have similar bindingproperties as the reference nucleic acid and are metabolized in a mannersimilar to naturally occurring nucleotides. Unless otherwise indicated,a particular nucleic acid sequence also implicitly encompassesconservatively modified variants thereof (e.g., degenerate codonsubstitutions) and complementary sequences and as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions canbe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al (1991) Nucleic Acid Res 19:5081;Ohtsuka et al. (1985) J Biol Chem 260:2605-2608; Rossolini et al. (1994)Mol Cell Probes 8:91-98). The terms “nucleic acid” or “nucleic acidsequence” can also be used interchangeably with gene, open reading frame(ORF), cDNA, mRNA, siRNA, microRNA, and the like.

The terms “small interfering RNA,” “short interfering RNA,” “smallhairpin RNA” “siRNA,” and “shRNA” are used interchangeably herein torefer to any nucleic acid molecule capable of mediating RNA interference(RNAi) or gene silencing. See, e.g., Bass, Nature 411:428-429, 2001;Elbashir et al, Nature 411:494-498, 2001a; and PCT InternationalPublication Nos. WO 00/44895, WO 01/36646, WO 99/32619, WO 00/01846, WO01/29058, WO 99/07409, and WO 00/44914. In one embodiment, the siRNA cancomprise a double stranded polynucleotide molecule comprisingcomplementary sense and antisense regions, wherein the antisense regioncomprises a sequence complementary to a region of a target nucleic acidmolecule. In another embodiment, the siRNA can comprise a singlestranded polynucleotide having self-complementary sense and antisenseregions, wherein the antisense region comprises a sequence complementaryto a region of a target nucleic acid molecule. In yet anotherembodiment, the siRNA can comprise a single stranded polynucleotidehaving one or more loop structures and a stem comprisingself-complementary sense and antisense regions, wherein the antisenseregion comprises a sequence complementary to a region of a targetnucleic acid molecule, and wherein the polynucleotide can be processedeither in vivo or in vitro to generate an active siRNA capable ofmediating RNAi. As used herein, siRNA molecules need not be limited tothose molecules containing only RNA, but further encompass chemicallymodified nucleotides and non-nucleotides.

MicroRNAs are naturally occurring, small non-coding RNAs that are about17 to about 25 nucleotide bases (nt) in length in their biologicallyactive form, miRNAs post-transcriptionally regulate gene expression byrepressing target mRNA translation. It is thought that miRNAs functionas negative regulators, i.e. greater amounts of a specific miRNA willcorrelate with lower levels of target gene expression. There are threeforms of miRNAs existing in vivo, primary miRNAs (pri-miRNAs), prematuremiRNAs (pre-miRNAs), and mature miRNAs, Primary miRNAs (pri-miRNAs) areexpressed as stem-loop structured transcripts of about a few hundredbases to over 1 kb. The pri-miRNA transcripts are cleaved in the nucleusby an RNase II endonuclease called Drosha that cleaves both strands ofthe stem near the base of the stem loop. Drosha cleaves the RNA duplexwith staggered cuts, leaving a 5′ phosphate and 2 nt overhang at the 3′end. The cleavage product, the premature miRNA (pre-miRNA) is about 60to about 110 nt long with a hairpin structure formed in a fold-backmanner. Pre-miRNA is transported from the nucleus to the cytoplasm byRan-GTP and Exportin-5. Pre-miRNAs are processed further in thecytoplasm by another RNase II endonuclease called Dicer. Dicerrecognizes the 5′ phosphate and 3′ overhang, and cleaves the loop off atthe stem-loop junction to form miRNA duplexes. The miRNA duplex binds tothe RNA-induced silencing complex (RISC), where the antisense strand ispreferentially degraded and the sense strand mature miRNA directs RISCto its target site. It is the mature miRNA that is the biologicallyactive form of the miRNA and is about 17 to about 25 nt in length.

In some embodiments, the nucleic acid molecules that are encapsulated orotherwise incorporated into a microvesicle composition of thepresently-disclosed subject matter are included in the microvesicles arepart of an expression vector. The term “expression vector” is usedinterchangeably herein with the terms “expression cassette” and“expression control sequence,” and is used to refer to a nucleic acidmolecule capable of directing expression of a particular nucleotidesequence in an appropriate host cell, comprising a promoter operativelylinked to the nucleotide sequence of interest which is operativelylinked to termination signals. It also typically comprises sequencesrequired for proper translation of the nucleotide sequence. The codingregion usually encodes a polypeptide of interest but can also encode afunctional RNA of interest, for example antisense RNA or anon-translated RNA, in the sense or antisense direction, or a non-codingRNA (ncRNA) such as a small or long ncRNA. The expression vectorcomprising the nucleotide sequence of interest can be chimeric, meaningthat at least one of its components is heterologous with respect to atleast one of its other components. The expression vector can also be onethat is naturally occurring but has been obtained in a recombinant formuseful for heterologous expression. In some embodiments, the expressionvector is a mammalian expression vector that is capable of directingexpression of a particular nucleic acid sequence of interest in amammalian cell.

With respect to the plasma membrane coating the edible-plant derivedmicrovesicles, the phrase “coating the microvesicle” and variationsthereof, is used herein to refer to the covering, placement, and/orattachment of a plasma membrane to the lipid bilayer of an exemplarymicrovesicle of the presently-disclosed subject matter, or a portionthereof. In some embodiments, the covering of a microvesicle with aplasma membrane derived from an activated inflammatory cell, such as aleukocyte, is achieved by initially exposing a population ofinflammatory cells to a cell activation compound (e.g., Phorbol12-Myristate 13-Acetate (PMA), a calcium ionophore such as lonomycin, ora protein transport inhibitor) capable of inducing the expression ofvarious cytokines, chemokines, and/or chemokine receptors. After aperiod of incubating the inflammatory cells with such an activationcompound, the inflammatory cells are then lysed and homogenized, and theplasma membranes are collected by sucrose gradient densitycentrifugation. After collecting the plasma membranes, the membranes aresubsequently sonicated to form vesicles. Such vesicles can then becombined with the microvesicles described herein (e.g., microvesiclesencapsulating a therapeutic agent) and co-extruded through a membrane tothereby coat the microvesicles with the plasma membranes derived fromthe inflammatory cells. In this regard, and without wishing to be boundby any particular, theory, it is believed that by virtue of using plasmamembranes deri ved from activated inflammatory cells, the resultingmicrovesiele compositions include a plasma membrane coating having oneor more chemokine receptor or other targeting moieties that are capableof homing or otherwise targeting the compositions to the site ofinflamed or diseased tissue.

In some embodiments of the presently disclosed subject matter, apharmaceutical composition is provided that comprises an edibleplant-derived microvesicle composition disclosed herein and apharmaceutical vehicle, carrier, or excipient. In some embodiments, thepharmaceutical composition is pharmaceudcally-acceptable in humans.Also, as described further below, in some embodiments, thepharmaceutical composition can be formulated as a therapeuticcomposition for delivery to a subject.

A pharmaceutical composition as described herein preferably comprises acomposition that includes pharmaceutical carrier such as aqueous andnon-aqueous sterile injection solutions that can contain antioxidants,buffers, bacteriostats, bactericidal antibiotics and solutes that renderthe formulation isotonic with the bodily fluids of the intendedrecipient; and aqueous and non-aqueous sterile suspensions, which caninclude suspending agents and thickening agents. The pharmaceuticalcompositions used can take such forms as suspensions, solutions oremulsions in oily or aqueous vehicles, and can contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.Additionally, the formulations can be presented in unit-dose ormulti-dose containers, for example sealed ampoules and vials, and can bestored in a frozen or freeze-dried or room temperature (lyophilized)condition requiring only the addition of sterile liquid carrierimmediately prior to use.

In some embodiments, solid formulations of the compositions for oraladministration can contain suitable carriers or excipients, such as cornstarch, gelatin, lactose, acacia, sucrose, microcrystalline cellulose,kaolin, mannitol, dicalcium phosphate, calcium carbonate, sodiumchloride, or alginic acid. Disintegrators that can be used include, butare not limited to, microcrystalline cellulose, corn starch, sodiumstarch glycolate, and alginic acid. Tablet binders that can be usedinclude acacia, methylcellulose, sodium carboxymethylcellulose,polyvinylpyrrolidone, hydroxypropyl methylcellulose, sucrose, starch,and ethylcellulose. Lubricants that can be used include magnesiumstearates, stearic acid, silicone fluid, talc, waxes, oils, andcolloidal silica. Further, the solid formulations can be uncoated orthey can be coated by known techniques to delay disintegration andabsorption in the gastrointestinal tract and thereby provide asustained/extended action over a longer period of time. For example,glyceryl monostearate or glyceryl distearate can be employed to providea sustained-/extended-release formulation. Numerous techniques forformulating sustained release preparations are known to those ofordinary skill in the art and can be used in accordance with the presentinvention, including the techniques described in the followingreferences; U.S. Pat. Nos. 4,891,223; 6,004,582; 5,397,574; 5,419,917;5,458,005; 5,458,887; 5,458,888; 5,472,708; 6,106,862; 6,103,263;6,099,862; 6,099,859; 6,096,340; 6,077,541; 5,916,595; 5,837,379;5,834,023; 5,885,616; 5,456,921; 5,603,956; 5,512,297; 5,399,362;5,399,359; 5,399,358; 5,725,883; 5,773,025; 6,110,498; 5,952,004;5,912,013; 5,897,876; 5,824,638; 5,464,633; 5,422,123; and 4,839,177;and WO 98/47491, each of which is incorporated herein by this reference.

Liquid preparations for oral administration can take the form of, forexample, solutions, syrups or suspensions, or they can be presented as adry product for constitution with water or other suitable vehicle beforeuse. Such liquid preparations can be prepared by conventional techniqueswith pharmaceutically-acceptable additives such as suspending agents(e.g., sorbitol syrup, cellulose derivatives or hydrogenated ediblefats); emulsifying agents (e.g. lecithin or acacia); non-aqueousvehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionatedvegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The preparations can alsocontain buffer salts, flavoring, coloring and sweetening agents asappropriate. Preparations for oral administration can be suitablyformulated to give controlled release of the active compound. For buccaladministration the compositions can take the form of capsules, tabletsor lozenges formulated in conventional manner.

Various liquid and powder formulations can also be prepared byconventional methods for inhalation into the lungs of the subject to betreated or for intranasal administration into the nose and sinuscavities of a subject to be treated. For example, the compositions canbe conveniently delivered in the form of an aerosol spray presentationfrom pressurized packs or a nebulizer, with the use of a suitablepropellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas.Capsules and cartridges of, for example, gelatin for use in an inhaleror insufflator may be formulated containing a powder mix of the desiredcompound and a suitable powder base such as lactose or starch.

The compositions can also be formulated as a preparation forimplantation or injection. Thus, for example, the compositions can beformulated with suitable polymeric or hydrophobic materials (e.g., as anemulsion in an acceptable oil) or ion exchange resins, or as sparinglysoluble derivatives (e.g., as a sparingly soluble salt).

Injectable formulations of the compositions can contain various carrierssuch as vegetable oils, dimethylacetamide, dimethylformamide, ethyllactate, ethyl carbonate, isopropyl myristate, ethanol, polyols(glycerol, propylene glycol, liquid polyethylene glycol), and the like.For intravenous injections, water soluble versions of the compositionscan be administered by the drip method, whereby a formulation includinga pharmaceutical composition of the presently-disclosed subject matterand a physiologically-acceptable excipient is infused.Physiologically-acceptable excipients can include, for example, 5%dextrose, 0.9% saline, Ringer's solution or other suitable excipients.Intramuscular preparations, e.g., a sterile formulation of a suitablesoluble salt form of the compounds, can be dissolved and administered ina pharmaceutical excipient such as Water-for-Injection, 0.9% saline, or5% glucose solution. A suitable insoluble form of the composition can beprepared and administered as a suspension in an aqueous base or apharmaceutically-acceptable oil base, such as an ester of a long chainfatty acid, (e.g., ethyl oleate).

In addition to the formulations described above, the microvesiclecompositions of the presently-disclosed subject matter can also beformulated as rectal compositions, such as suppositories or retentionenemas, e.g., containing conventional suppository bases such as cocoabutter or other glycerides. Further, the exosomal compositions can alsobe formulated as a depot preparation by combining the compositions withsuitable polymeric or hydrophobic materials (for example as an emulsionin an acceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt.

Further provided, in some embodiments of the presently-disclosed subjectmatter, are methods for treating an inflammatory disorder or a cancer.In some embodiments, a method for treating an inflammatory disorder isprovided that comprises administering to a subject in need thereof aneffective amount of a microvesicle composition of thepresently-disclosed subject matter.

As used herein, the terms “treatment” or “treating” relate to anytreatment of a condition of interest (e.g., an inflammatory disorder ora cancer), including but not limited to prophylactic treatment andtherapeutic treatment. As such, the terms “treatment” or “treating”include, but are not limited to: preventing a condition of interest orthe development of a condition of interest; inhibiting the progressionof a condition of interest; arresting or preventing the furtherdevelopment of a condition of interest; reducing the severity of acondition of interest; ameliorating or relieving symptoms associatedwith a condition of interest; and causing a regression of a condition ofinterest or one or more of the symptoms associated with a condition ofinterest.

As used herein, the term “inflammatory disorder” includes diseases ordisorders which are caused, at least in part, or exacerbated, byinflammation, which is generally characterized by increased blood flow,edema, activation of immune cells (e.g., proliferation, cytokineproduction, or enhanced phagocytosis), heat, redness, swelling, painand/or loss of function in the affected tissue or organ. The cause ofinflammation can be due to physical damage, chemical substances,micro-organisms, tissue necrosis, cancer, or other agents or conditions.

Inflammatory disorders include acute inflammatory disorders, chronicinflammatory disorders, and recurrent inflammatory disorders. Acuteinflammatory disorders are generally of relatively short duration, andlast for from about a few minutes to about one to two days, althoughthey can last several weeks. Characteristics of acute inflammatorydisorders include increased blood flow, exudation of fluid and plasmaproteins (edema) and emigration of leukocytes, such as neutrophils.Chronic inflammatory disorders, generally, are of longer duration, e.g.,weeks to months to years or longer, and are associated histologicallywith the presence of lymphocytes and macrophages and with proliferationof blood vessels and connective tissue. Recurrent inflammatory disordersinclude disorders which recur after a period of time or which haveperiodic episodes. Some inflammatory disorders fall within one or morecategories. Exemplary inflammatory disorders include, but are notlimited to atherosclerosis; arthritis; inflammation-promoted cancers;asthma; autoimmune uveitis; adoptive immune response; dermatitis;multiple sclerosis; diabetic complications; osteoporosis; Alzheimer'sdisease; cerebral malaria; hemorrhagic fever; autoimmune disorders; andinflammatory bowel disease. In some embodiments, the inflammatorydisorder is selected from the group consisting of sepsis, septic shock,colitis, colon cancer, and arthritis.

For administration of a therapeutic composition as disclosed herein(e.g., an edible plant-derived microvesicle encapsulating a therapeuticagent), conventional methods of extrapolating human dosage based ondoses administered to a murine animal model can be carried out using theconversion factor for converting the mouse dosage to human dosage: DoseHuman per kg=Dose Mouse per kg×12 (Freireich, et al., (1966) CancerChemother Rep. 50:219-244). Doses can also be given in milligrams persquare meter of body surface area because this method rather than bodyweight achieves a good correlation to certain metabolic and excretionaryfunctions. Moreover, body surface area can be used as a commondenominator for drug dosage in adults and children as well as indifferent animal species as described by Freireich, et al. (Freireich etal., (1966) Cancer Chemother Rep. 50:219-244). Briefly, to express amg/kg dose in any given species as the equivalent mg/sq m dose, multiplythe dose by the appropriate km factor. In an adult human, 100 mg/kg isequivalent to 100 mg/kg×37 kg/sq m=3700 mg/m².

Suitable methods for administering a therapeutic composition inaccordance with the methods of the presently-disclosed subject matterinclude, but are not limited to, systemic administration, parenteraladministration (including intravascular, intramuscular, and/orintraarterial administration), oral delivery, buccal delivery, rectaldelivery, subcutaneous administration, intraperitoneal administration,inhalation, intratracheal installation, surgical implantation,transdermal delivery, local injection, intranasal delivery, andhyper-velocity injection/bombardment. Where applicable, continuousinfusion can enhance drug accumulation at a target site (see, e.g., U.S.Pat. No. 6,180,082).

Regardless of the route of administration, the compositions of thepresently-disclosed subject matter are typically administered in amounteffective to achieve the desired response. As such, the term “effectiveamount” is used herein to refer to an amount of the therapeuticcomposition (e.g., a microvesicle encapsulating a therapeutic agent, anda pharmaceutically vehicle, carrier, or excipient) sufficient to producea measurable biological response (e.g., a decrease in inflammation).Actual dosage levels of active ingredients in a therapeutic compositionof the present invention can be varied so as to administer an amount ofthe active compound(s) that is effective to achieve the desiredtherapeutic response for a particular subject and/or application. Ofcourse, the effective amount in any particular case will depend upon avariety of factors including the activity of the therapeuticcomposition, formulation, the route of administration, combination withother drugs or treatments, severity of the condition being treated, andthe physical condition and prior medical history of the subject beingtreated. Preferably, a minimal dose is administered, and the dose isescalated in the absence of dose-limiting toxicity to a minimallyeffective amount. Determination and adjustment of a therapeuticallyeffective dose, as well as evaluation of when and how to make suchadjustments, are known to those of ordinary skill in the art.

For additional guidance regarding formulation and dose, see U.S. Pat.Nos. 5,326,902; 5,234,933; PCT International Publication No. WO93/25521; Berkow et al., (1997) The Merck Manual of Medical Information,Home ed, Merck Research Laboratories, Whitehouse Station, N.J.; Goodmanet al., (1996) Goodman & Gilman's the Pharmacological Basis ofTherapeutics, 9th ed. McGraw-Hill Health Professions Division, New York;Ebadi, (1998) CRC Desk Reference of Clinical Pharmacology, CRC Press,Boca Raton, Fla.; Katzung, (2001) Basic & Clinical Pharmacology, 8th ed.Lange Medical Books/McGraw-Hill Medical Pub. Division, New York;Remington et al., (1975) Remington's Pharmaceutical Sciences, 15th ed.Mack Pub. Co., Easton, Pa.; and Speight et al., (1997) Avery's DrugTreatment: A Guide to the Properties, Choice, Therapeutic Use andEconomic Value of Drugs in Disease Management, 4th ed. AdisInternational, Auckland/Philadelphia; Duch et al., (1998) Toxicol. Lett.100-101:255-263.

In some embodiments of the therapeutic methods disclosed herein,administering an edible plant-derived microvesicle composition of thepresently-disclosed subject matter reduces an amount of an inflammatorycytokine in a subject. In some embodiments, the inflammatory cytokinecan be interleukin-1β (IL-1β), tumor necrosis factor-alpha (TNF-α),interferon-γ (IFN-γ), or interleukin-6 (IL-6).

Various methods known to those skilled in the art can be used todetermine a reduction in the amount of inflammatory cytokines in asubject. For example, in certain embodiments, the amounts of expressionof an inflammatory cytokine in a subject can be determined by probingfor mRNA of the gene encoding the inflammatory cytokine in a biologicalsample obtained from the subject (e.g., a tissue sample, a urine sample,a saliva sample, a blood sample, a serum sample, a plasma sample, orsub-fractions thereof) using any RNA identification assay known to thoseskilled in the art. Briefly, RNA can be extracted from the sample,amplified, converted to cDNA, labeled, and allowed to hybridize withprobes of a known sequence, such as known RNA hybridization probesimmobilized on a substrate, e.g., array, or microarray, or quantitatedby real time PCR (e.g., quantitative real-time PCR, such as availablefrom Bio-Rad Laboratories, Hercules, Calif.). Because the probes towhich the nucleic acid molecules of the sample are bound are known, themolecules in the sample can be identified. In this regard, DNA probesfor one or more of the mRNAs encoded by the inflammatory genes can beimmobilized on a substrate and provided for use in practicing a methodin accordance with the presently-disclosed subject matter.

With further regard to determining levels of inflammatory cytokines insamples, mass spectrometry and/or immunoassay devices and methods canalso be used to measure the inflammatory cytokines in samples, althoughother methods can also be used and are well known to those skilled inthe art. See, e.g., U.S. Pat. Nos. 6,143,576; 6,113,855; 6,019,944;5,985,579; 5,947,124; 5,939,272; 5,922,615; 5,885,527; 5,851,776;5,824,799; 5,679,526; 5,525,524; and 5,480,792, each of which is herebyincorporated by reference in its entirety. Immunoassay devices andmethods can utilize labeled molecules in various sandwich, competitive,or non-competitive assay formats, to generate a signal that is relatedto the presence or amount of an analyte of interest. Additionally,certain methods and devices, such as biosensors and opticalimmunoassays, can be employed to determine the presence or amount ofanalytes without the need for a labeled molecule. See, e.g., U.S. Pat.Nos. 5,631,171; and 5,955,377, each of which is hereby incorporated byreference in its entirety.

Any suitable immunoassay can be utilized, for example, enzyme-linkedimmunoassays (ELISA), radioimmunoassays (RIAs), competitive bindingassays, and the like. Specific immunological binding of the antibody tothe inflammatory molecule can be detected directly or indirectly. Directlabels include fluorescent or luminescent tags, metals, dyes,radionucieotides, and the like, attached to the antibody. Indirectlabels include various enzymes well known in the art, such as alkalinephosphatase, horseradish peroxidase and the like.

The use of immobilized antibodies or fragments thereof specific for theinflammatory molecules is also contemplated by the present invention.The antibodies can be immobilized onto a variety of solid supports, suchas magnetic or chromatographic matrix particles, the surface of an assayplate (such as microliter wells), pieces of a solid substrate material(such as plastic, nylon, paper), and the like. An assay strip can beprepared by coating the antibody or a plurality of antibodies in anarray on a solid support. This strip can then be dipped into the testbiological sample and then processed quickly through washes anddetection steps to generate a measurable signal, such as for example acolored spot.

Mass spectrometry (MS) analysis can be used, either alone or incombination with other methods (e.g., immunoassays), to determine thepresence and/or quantity of an inflammatory molecule in a subject.Exemplary MS analyses that can be used in accordance with the presentinvention include, but are not limited to: liquid chromatography-massspectrometry (LC-MS); matrix-assisted laser desorption/ionizationtime-of-flight MS analysis (MALDI-TOF-MS), such as for exampledirect-spot MALDI-TOF or liquid chromatography MALDI-TOF massspectrometry analysis; electrospray ionization MS (ESI-MS), such as forexample liquid chromatography (LC) ESI-MS; and surface enhanced laserdesorption/ionization time-of-flight mass spectrometry analysis(SELDI-TOF-MS). Each of these types of MS analysis can be accomplishedusing commercially-available spectrometers, such as, for example, triplequadropole mass spectrometers. Methods for utilizing MS analysis todetect the presence and quantity of peptides, such as inflammatorycytokines, in biological samples are known in the art. See, e.g., U.S.Pat. Nos. 6,925,389; 6,989,100; and 6,890,763 for further guidance, eachof which are incorporated herein by this reference.

With still further regard to the various therapeutic methods describedherein, although certain embodiments of the methods disclosed hereinonly call for a qualitative assessment (e.g., the presence or absence ofthe expression of an inflammatory cytokine in a subject), otherembodiments of the methods call for a quantitative assessment (e.g., anamount of increase in the level of an inflammatory cytokine in asubject). Such quantitative assessments can be made, for example, usingone of the above mentioned methods, as will be understood by thoseskilled in the art.

The skilled artisan will also understand that measuring a reduction inthe amount of a certain feature (e.g., cytokine levels) or animprovement in a certain feature (e.g., inflammation) in a subject is astatistical analysis. For example, a reduction in an amount ofinflammatory cytokines in a subject can be compared to control level ofinflammatory cytokines, and an amount of inflammatory cytokines of lessthan or equal to the control level can be indicative of a reduction inthe amount of inflammatory cytokines, as evidenced by a level ofstatistical significance. Statistical significance is often determinedby comparing two or more populations, and determining a confidenceinterval and/or a p value. See, e.g., Dowdy and Wearden, Statistics forResearch, John Wiley & Sons, New York, 1983, incorporated herein byreference in its entirety. Preferred confidence intervals of the presentsubject matter are 90%, 95%, 97.5%, 98%, 99%, 99.5%, 99.9% and 99.99%,while preferred p values are 0.1, 0.05, 0.025, 0.02, 0.01, 0.005, 0.001,and 0.0001.

Still further provided, in some embodiments, are methods for treating acancer. In some embodiments, a method for treating a cancer is providedthat comprises administering to a subject in need thereof an effectiveamount of an edible-plant derived microvesicle composition of thepresently-disclosed subject matter (i.e., where a plasma membrane-coatedmicrovesicle encapsulates a therapeutic agent). In some embodiments, thetherapeutic agent encapsulated within the plasma membrane-coatedmicrovesicle and used to treat the cancer is selected from aphytochemical agent and a chemotherapeutic agent, as such agents havebeen found to be particularly useful in the treatment of cancer. As usedherein, the term “cancer” refers to all types of cancer or neoplasm ormalignant tumors found in animals, including leukemias, carcinomas,melanoma, and sarcomas.

By “leukemia” is meant broadly progressive, malignant diseases of theblood-forming organs and is generally characterized by a distortedproliferation and development of leukocytes and their precursors in theblood and bone marrow. Leukemia diseases include, for example, acutenonlymphocytic leukemia, chronic lymphocytic leukemia, acutegranulocytic leukemia, chronic granulocytic leukemia, acute promyelocyteleukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemicleukemia, basophylic leukemia, blast cell leukemia, bovine leukemia,chronic myelocytic leukemia, leukemia cutis, embryonal leukemia,eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia, hemoblasticleukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cellleukemia, acute monocytic leukemia, leukopenic leukemia, lymphaticleukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenousleukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cellleukemia, megakaryocyte leukemia, micromyeloblastic leukemia, monocyticleukemia, myeloblastic leukemia, myelocytic leukemia, myeloidgranulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasmacell leukemia, plasmacytic leukemia, promyelocyte leukemia, Rieder cellleukemia, Schilling's leukemia, stem cell leukemia, subleukemicleukemia, and undifferentiated cell leukemia.

The term “carcinoma” refers to a malignant new growth made up ofepithelial cells tending to infiltrate the surrounding tissues and giverise to metastases. Exemplary carcinomas include, for example, acinarcarcinoma, acinous carcinoma, adenocystie carcinoma, adenoid cysticcarcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolarcarcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinomabasocellulare, basaloid carcinoma, basosquamous cell carcinoma,bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogeniccarcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorioniccarcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma,cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum,cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma,carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiennoidcarcinoma, carcinoma epitheliale adenoides, exophytic carcinoma,carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma,gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare,glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma,hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma,hyaline carcinoma, hypemephroid carcinoma, infantile embryonalcarcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelialcarcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cellcarcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatouscarcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullarycarcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma,carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma,carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes,nasopharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans,osteoid carcinoma, papillary carcinoma, periportal carcinoma,preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma,renal cell carcinoma of kidney, reserve cell carcinoma, carcinomasarcomatodes, schneiderian carcinoma, scirrhous carcinoma, carcinomascroti, signet-ring cell carcinoma, carcinoma simplex, small-cellcarcinoma, solanoid carcinoma, spheroidal cell carcinoma spindle cellcarcinoma, carcinoma spongiosum, squamous carcinoma, squamous cellcarcinoma, string carcinoma, carcinoma telangiectaticum, carcinomatelangiectodes, transitional cell carcinoma, carcinoma tuberosum,tuberous carcinoma, verrucous carcinoma, and carcinoma villosum.

The term “sarcoma” generally refers to a tumor which is made up of asubstance like the embryonic connective tissue and is generally composedof closely packed cells embedded in a fibrillar or homogeneoussubstance. Sarcomas include, for example, chondrosarcoma, fibrosarcoma,lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abemethy'ssarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma,ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, choriocarcinoma, embryonal sarcoma, Wilns' tumor sarcoma, endometrial sarcoma,stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma,giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathicmultiple pigmented hemorrhagic sarcoma, immuaoblastic sarcoma of Bcells, lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma,Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma,malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocyticsarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, andtelangiectaltic sarcoma.

The term “melanoma” is taken to mean a tumor arising from the melanocytesystem of the skin and other organs. Melanomas include, for example,acral-lentiginous melanoma, amelanotic melanoma, benign juvenilemelanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma,juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodularmelanoma subungal melanoma, and superficial spreading melanoma.

Additional cancers include, for example, Hodgkin's Disease,Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast cancer,ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis,primary macroglobulinemia, small-cell lung tumors, primary brain tumors,stomach cancer, colon cancer, malignant pancreatic insulanoma, malignantcarcinoid, premalignant skin lesions, testicular cancer, lymphomas,thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tractcancer, malignant hypercalcemia, cervical cancer, endometrial cancer,and adrenal cortical cancer. In some embodiments, the cancer is selectedfrom the group consisting of skin cancer, head and neck cancer, coloncancer, breast cancer, brain cancer, and lung cancer.

As used herein, the term “subject” includes both human and animalsubjects. Thus, veterinary therapeutic uses are provided in accordancewith the presently disclosed subject matter. As such, thepresently-disclosed subject matter provides for the treatment of mammalssuch as humans, as well as those mammals of importance due to beingendangered, such as Siberian tigers; of economic importance, such asanimals raised on farms for consumption by humans; and/or animals ofsocial importance to humans, such as animals kept as pets or in zoos.Examples of such animals include, but are not limited to: carnivoressuch as cats and dogs; swine, including pigs, hogs, and wild boars;ruminants and/or ungulates such as cattle, oxen, sheep, giraffes, deer,goats, bison, and camels; and horses. Also provided is the treatment ofbirds, including the treatment of those kinds of birds that areendangered and/or kept in zoos, as well as fowl, and more particularlydomesticated fowl, i.e., poultry, such as turkeys, chickens, ducks,geese, guinea fowl, and the like, as they are also of economicimportance to humans. Thus, also provided is the treatment of livestock,including, but not limited to, domesticated swine, ruminants, ungulates,horses (including race horses), poultry, and the like.

The practice of the presently-disclosed subject matter can employ,unless otherwise indicated, conventional techniques of cell biology,cell culture, molecular biology, transgenic biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature. See e.g..Molecular Cloning A Laboratory Manual (1989), 2nd Ed., ed. by Samhrook,Fritsch and Maniatis, eds., Cold Spring Harbor Laboratory Press,Chapters 16 and 17; U.S. Pat. No. 4,683,195; DNA Cloning, Volumes I andII, Glover, ed., 1985; Oligonucleotide Synthesis. M. J. Gait, ed., 1984;Nucleic Acid Hybridization, D. Hames & S. J. Higgins, eds., 1984;Transcription and Translation, B. D. Hames & S. J. Higgins, eds., .1984;Culture Of Animal Cells, R. I. Freshney, Alan R. Liss, Inc., 1987;Immobilized Cells And Enzymes, IRL Press, 1986; Perbal (1984), APractical Guide To Molecular Cloning; See Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells,J. H. Miller and M. P. Calos, eds., Cold Spring Harbor Laboratory, 1987;Methods In Enzymology, Vols. 154 and 155, Wu et al., eds., AcademicPress Inc., N.Y.; Immunochemical Methods In Cell And Molecular Biology(Mayer and Walker, eds., Academic Press, London, 1987; Handbook OfExperimental Immunology, Volumes I-IV, D. M. Weir and C. C. Blackwell,eds., 1986.

The presently-disclosed subject matter is further illustrated by thefollowing specific but non-limiting examples.

EXAMPLES

Inflammation is a hallmark of numerous diseases. Activated immune cellsare intrinsically capable of homing to inflammatory sites. Using threeinflammatory driven disease mouse models, the following studiesdemonstrated that grapefruit-derived nanovectors (GNVs) coated withinflammatory chemokine receptor enriched membranes of activatedleukocytes (IGNVs) are enhanced for homing to inflammatory tissues.Blocking CXCR1 and CXCR2 on the IGNVs significantly inhibited IGNVshoming to the inflammatory tissue. The therapeutic potential of IGNVswas further demonstrated by enhancing the chemotherapeutic effect asshown by inhibition of tumor growth in two tumor models and inhibitingthe inflammatory effects of DSS induced mouse colitis. The fact thatIGNVs are capable of homing to inflammatory tissue and that there is anoverexpression of chemokines in diseased human tissue provides supportfor using IGNVs for the more directed delivery of therapeutic agents toinflammatory sites and the use of IGNVs as personalized medicine fortreatment of inflammatory related diseases.

Materials and Methods

Mice. C57BL/6j mice, BALB/c mice, and C.129S2(B6)-Cxcr2^(umlMwm)/J mice6-8 weeks of age were obtained from Jackson Laboratories. All animalprocedures were approved by the University of Louisville InstitutionalAnimal Care and Use Committee.

Cell culture. The mouse T lymphoma EL4 cells, mouse 4T1, 4TO7 breastcancer cell lines, mouse NMuMG mammary gland epithelial cells, CT26colon cancer and human umbilical vein endothelial cells (HUVECs) werepurchased from ATCC. CT26 cells were cultured in RPMI 1640 media; EL4,4T1 and 4TO7 cells were maintained in DMEM media supplemented with 10%heat-inactivated FBS. HUVECs were cultured in complete endothelial cellgrowth medium (ECGM; Promocell #C-22010). NMuMG cells were cultured incomplete DMEM media supplemented with 10 μg/ml insulin. All cells weremaintained in a humidified CO₂ incubator at 37° C.

Reagents and antibodies. Doxorubicin, curcumin, phorbol 12-myristate13-acetate (PMA), lipopolysaccharide (LPS, Escherichia coli 0111:B4),PKH67, PKH26, and Fluorescent Cell Linker kits were purchased fromSigma-Aldrich (St Louis, Mo., USA). Commercial DOX-NP™ (300102S) and itscontrol liposomes were purchased from Avanti Polar Lipids Inc.(Alabaster, Ala., USA). Proteinase inhibitor cocktail tablets wereobtained from Roche Diagnostics GmbH (Mannheim, Germany). DiR dye wasobtained from Life Technologies (NY, USA). Dextran sodium sulfate (DSS,MW: 36,000-50,000) was obtained from MP Biomedicals, LLC (Santa Ana,Calif.). Infinity™ ALT (GPT) and AST (GOT) liquid stable reagents werepurchased from Thermo Scientific (Pittsburgh, Pa., USA). TheLuminescence ATP detection system was obtained from PerkinElmer(Waltham, Mass., USA). Recombinant mouse chemokines CXCL1, CXCL2, CXCL9,CXCL10, CXCL11, CCL2, and CCL5 were purchased from Biolegend (San Diego,Calif., USA). Fluorescent conjugated antibodies against mouse or humanCCR3, CCR4, CCR5, CCR7, CCR9, CXCR2, CXCR3, CXCR7, antibodies againstmouse CD3, F4/80, CD11b, CD11c, CD19, Ly6G and Gr1 were purchased fromeBiosicence (San Diego, Calif., USA), Anti-human CXCL1, CXCL10, CCL2 andCCL5 antibodies were obtained from Lifespan Biosciences, Inc. (Seattle,Wash., USA). Anti-mouse CXCL1, CXCL10, CCL2 and CCL5 antibodies andCCL5, CXCL10 ELISA kits were purchased from Biolegend (San Diego,Calif., USA). Alexa Fluor 488 nm and 647 nm conjugated secondaryantibodies were obtained from Life Technologies (NY, USA). The HumanCCL2 ELISA kit was purchased from eBiosicence.

Plasma membrane isolation and purification. Plasma membranes wereisolated and purified using a method as described previously. In brief,mouse lymphoma cell line EL4 cells were cultured with/without PMAstimulation (500 ng/ml) for 12 h. Cells (2×10⁸) were then collected andcentrifuged at 500×g for 10 min at 4° C. The cell pellets wereresuspended in 1 ml of homogenization buffer at a final concentration of10 mM Tris HCL, 25 mM D-sucrose, 1 mM MgCl₂, 1 mM KCl, 10 μg/ml RNase,10 μg/ml DNase and 1× proteinase inhibitor cocktail. The cell suspensionwas homogenized on ice by 100 passes using a hand-held Douncehomogenizer. The supernatant was collected after centrifugation at 500×gfor 10 min. For further purification, collected supernatants weresubjected to a discontinuous sucrose density gradient centrifugation at28,000 g for 45 min at 4° C. on a 30%, 40% and 55% sucrose in a 0.9%saline solution. For plasma membrane isolation from leukocytes of miceor human peripheral blood, anticoagulant treated peripheral blood wascollected and centrifuged at 3000×g for 10 min at 4° C. Leukocytes werecollected and the red blood cells were lysed by incubation with ACKlysis buffer (NH₄Cl 8.024 g/L, KHCO₃ 1 g/L, EDTA-2Na 3.722 mg/L) for 5mm at 22° C. The procedure for lysis of red blood cells was repeatedonce. The leukocytes were then cultured in RPMI 1640 with LPS (100ng/ml) for 12 h before the plasma membranes from leukocytes of mice orhuman with/without LPS stimulation were isolated and purified using adiscontinuous sucrose density gradient centrifugation method asdescribed above.

Preparation of plasma membram-derived vesicles. The plasma membranespurified from EL4 cells and mice or human peripheral blood leucocyteswere sonicated in a glass vial with 200 μl ddH₂O for 10 min using aFS30D bath sonicatar (Fisher Scientific). The resulting vesicles weresubsequently extruded through 100 nm polycarbonate porous membranesusing an Avanti mini extruder (Avanti Polar Lipids).

Preparation of plasma membrane-coated GNVs. Grapefruit lipid derivednanoparticles (GNVs) were prepared according to the protocol asdescribed previously. To prepare the plasma membrane coated GNVs, 400nmol of GNVs were mixed with plasma membrane-derived vesicles (fromapproximately 5×10⁵ cells) and extruded 20 times through a 200 nmpolycarbonate porous membrane using an Avanti mini extruder. The GNVscoated with EL4 derived or mouse or human leukocyte-derived plasmamembranes were then referred to as pseudo-inflammatory GNVs (IGNVs).

Phosphorus quantification. Phosphate in GNVs was quantified using astandard phosphorus solution (0.65 mM, P3869-25 ml from Sigma). First,different amounts of phosphate standard solutions (50, 25, 10, 5 and 0nmol) were prepared in 100 μl ddH₂O, then 30 μl Mg(NO₃)₂ was added, andthe mixture was heated by flame until dry. The dried sample wasdissolved in 300 μl HCl and heated at 100° C. for 15 min, cooled, andcentrifuged at 1000 rpm for 2 min, 700 μl of the reaction buffer (1 partof 10% ascobic acid and 6 parts 0.42% ammonium molybdate in 1 N H₂SO₄)were added and mixture incubated at 45° C. for 20 min. The absorptionwas read at OD₂₈₀.

Critical GNV miceller concentration (cmc). The critical micelleconcentration (CMC) is approximately defined as the lipid monomerconcentration at which appreciable amounts of micellar aggregates firstbegin to appear at equilibrium. Because GNVs are not monomers and aremade from a number of lipids extracted from grapefruit nanoparticles, itwas difficult to determine the CMC for each of grapefruit nanoparticlederived lipids that contribute to the formation of the GNVs. Therefore,unlike the common approach used for determination of CMC, an alternativeapproach was taken to determine the concentration of total GNV lipidsabove which GNVs form. In brief, total lipids extracted from grapefruitnanoparticle were determined using the phosphate assay as describedabove. Different volume of total lipids (0-100 μl, 200 uM in stock) inchloroform were pipetted into glass tubes, and the solvent was removedunder a stream of nitrogen, and the lipid films were subsequentlymaintained under vacuum condition for 2 h. The dry lipid films werehydrated at 60° C. for 30 min in 20 mM HEPES buffer (pH 7.0). After abath-sonication (FS60 bath sonicator, Fisher Scientific, Pittsburg, Pa.)for 5 min, an equal volume of HEPES buffer (pH 7.0) was added andsonicated for another 5 min.

Size distribution and Zeta potential analysis. Size distribution andZeta potential of particles were analyzed by a Zetasizer nano ZS(Malvern Instruments Ltd., Southborough, Mass.). To measure size,particle samples were diluted in PBS and dispersed by sonication for 5seconds before measuring. To determine the Zeta potential of particles,GNVs or plasma membrane coated GNVs were washed in ddH₂O bycentrifugation at 100,000×g for 45 min at 4° C. The samples wereresuspended in 1 ml ddH₂O for measuring the Zeta potential of theparticles.

FACS analysis of chemokine receptors, integrins on EL4 cells and IGNVs.To test the expression of chemokines receptors and integrins, collectedEL4 cells were washed, and blocked with Fc blocker (2.4G2) at 4° C. for15 min and then stained with PE labeled anti-CCR3, CCR4, CCR5, CCR7,CCR9, CXCR2, CXCR3, CXCR7, a4b7 and LFA-1 antibodies at 4° C. for 45min. The chemokine receptors and LFA-1 on the IGNVs were analyzedquantitatively using a FACS method as described previously. Briefly,GNVs or IGNVs were incubated with 4 mm-diameter aldehyde/sulfate latexbeads in 400 ml PBS containing 2% FCS for 30 min and the mixtures werethen washed and subsequently stained with PE labeled anti-CCR3, CCR4,CCR5, CCR7, CCR9, CXCR2, CXCR3, CXCR7 and LFA-1 antibodies at 4° C. for45 min. Since no α4β7 was detected on the EL4 cells; the levels of α4β7on the IGNVs was not analyzed. All samples were analyzed using an AccuriC6 flow cytometer.

Chemokine detection in skin and tumor tissues. Chemokines in normal skintissue, LPS induced inflammatory skin, and CT26 and 4T1 tumor tissueswere detected using Proteome Profiler™ antibody arrays (R&D System,Minneapolis, Minn., USA) according to the manufacturer's protocol.Briefly, tissues were excised and homogenized in PBS with 1× proteaseinhibitors and a final concentration of 1% Triton X-100. The sampleswere frozen at −80° C. After thawing, the supernatant of samples wascollected by centrifugation at 10,000×g for 5 min at 4° C. and the totalprotein was quantified using a NanoDrop 8000. After blocking for 1 h,the membranes were incubated with a mixture of reconstituted CytokineArray Panel A Detection Antibody Cocktail and the supernatant overnightat 4° C. After washing three times, the membranes were incubated withstreptavidin-HRP for 30 min at 22° C. Then, after washing three moretimes, the membranes were incubated with 1 ml of Chemi Regent Mix for1-2 min at 22° C. before exposing to X-ray film for 1-5 min.

Imaging the plasma membrane coated GNVs (IGNVs). The purified IGNVs wereprepared for electron microscopy using a conventional procedure andobserved using an FEI Tecnai F20 electron microscope operated at 200 kVat a magnification of 38,000× and defocus of 2.5 μm. Fresh prepared GNVswere also fixed and imaged as a control. Photomicrographs were takenusing a Gatan Ultrascan 4000 CCD camera.

To further confirm the co-localization of EL4 cell derived plasmamembrane with GNVs, PKH67 labeled membrane-derived vesicles were mixedwith PKH26 labeled GNVs and then extruded 15-20 times through a 200 nmpolycarbonate porous membrane using an Avanti mini extruder. Afterwashing at 100,000×g for 30 min, the particles were resuspended andincubated with 4T1 cells for 12 h in a 5% CO₂ incubator. The cells werethen washed 3 times with PBS, fixed with 2% PFA for 10 min at 22° C.,and permeabilized with 0.2% Triton X-100 for 2 min at 22° C. Afterwashing 3×, cells were stained with DAPI for 90 s. The co-localizationof EL4 cell derived plasma membrane with GNVs was examined using aconfocal microscope equipped with a digital image analysis system(Pixera, San Diego, Calif., USA).

Fluorescence resonance energy transfer (FRET) analysis. The DiO/DPAsystem has been used for detection of cytoplasmic membrane potentialchanges using the principle of fluorescence resonance energy transfer(FRET). This approach was adapted in the present studies by pairing thecommon tracer dye DiO (as a fluorescent donor) with dipicrylamine (DPA),a low-molecular-weight lipophilic anion, as a non-fluorescent acceptor.As a donor, DiO is a bright, nontoxic membrane label that permitsrepeated imaging of fluorescent labeled GNVs. However, if DiO is inclose proximity to the DPA dye labeled plasma membrane from activated Tcells, which enables fluorescence quenching, the result is nofluorescent signal detected. To test whether IGNVs are coated withplasma membrane from activated T cells, DiO (5 mM) labeled GNVs and DPA(5 mM) labeled EL4 cell membrane derived-vesicles were prepared bysonication using a method as described previously. The extrusion of GNVswith EL4 derived membrane vesicles was carried out by extrusion for 20times through a 200 nm polycarbonate porous membrane using an Avantimini extruder. The mixture of DiO (5 mM) labeled GNVs and DPA (5 mM)labeled EL4 cells membrane derived-vesicles without extrusion was usedas a control. Both samples were then diluted (1:5, 1:10, 1:20, 1:50,1:100, and 1:150) with ddH₂O and the fluorescent intensity of DiO dyewas measured by a fluorescent plate reader (Biotek HTS Multi-modeReaders). The non-quenched fluorescent signal, expressed as % offluorescent intensity, was determined as follows: [(fluorescentintensity of diluted sample—fluorescent intensity of undilutedsample)/fluorescent intensity of diluted sample]×100%. The resultsrepresent the mean of three independent experiments (error bars,standard errors of the means).

Transwell assay. HUVEC cells were seeded onto fibronectin-coated (7.5mg/cm²) Costar Transwell inserts (6.5 mm diameter and 5 mm pore size.Corning, Corning Incorporated, NY, USA) at a cell density of 5×10⁴cells/well. Chemokines were placed in the lower chamber. PKH26 labeledGNVs, IGNVs, chemokines or anti-LFA-1 antibody pre-incubated IGNVs wereadded into the upper chamber and incubated with HUVEC cells at 37° C.for 24 and 48 h. After incubation, the media from the lower chamber wascollected and diluted. The fluorescent intensity of PKH26 labeled GNVsor IGNVs was measured by a fluorescent plate reader (Biotek HTSMulti-mode Readers). The fluorescent intensity of PKH26 labeled GNVs orIGNVs, expressed as % of transwell efficiency of fluorescent intensityof PKH26 labeled GNVs or IGNVs, was determined as follows: (PKH26fluorescent intensity of the bottom well at the time of samples whichwere harvested/total PKH26 fluorescent intensity of PKH26 labeled GNVsor IGNVs added to the top well of the chamber at the beginning ofculture)×100%. The media from the lower chamber was collected andcentrifuged. Then the particles were resuspended in ddH2O, smeared ontoa slide and 4′,6-diamidino-2-phenylindole (DAPI) stained. The number ofHUVEC cell associated particles in the lower chamber was estimated bycounting 10 randomly selected fields (×20 magnification) using ImageJsoftware. None of the samples examined were positive for DAPI staining.

Hematoxylin and Eosin (H&E) and immunohistochemistry (IHC) staining.Livers, lungs, spleens, kidneys from BALB/c mice treated with PBS orIGNVs (200 nmol and 800 nmol, I.V. injection for three times) and colontissues from DSS fed mice were fixed overnight in 4% paraformaldehydeand embedded in paraffin; 5 μm sections of tissues were than stainedwith H&E.

Human breast cancer tissues and colon cancer tissues were collected inHuai'an First People's Hospital of China. All patients provided writteninformed consent. The use of human tissues in this study was approved bythe institutional review board of the Huai'an First People's Hospital ofChina and was conducted in accordance with international guidelines forthe use of human tissues. Paraffin embedded breast cancer, colon cancerand adjacent tissues sections (5 μm) were rehydrated and heated in anantigen retrieval solution for 45 min. Endogenous peroxidase activitywas inhibited by incubation with 3% hydrogen peroxide for 10 min at 22°C. and the non-specific sites were blocked with 5% BSA for 45 min. Thesectioned tissues on slides were then incubated with primary antibodies[(polyclonal anti-CXCL1 (0.15 μg/ml), polyclonal anti-CXCL10 (1 μg/ml),anti-CCL2 (1 μg/ml), and CCL5 (5 μg/ml)] overnight at 4° C. Sectionswere processed with appropriate biotinylated secondary antibody and astreptavidin biotin peroxidase amplification kit (Vectastain, VectorLaboratories, Burlingame, Calif.). The peroxidase reaction was finallydeveloped with diaminobenzidine (Dako) and sections were counter stainedwith Mayer's haematoxylin. Slides were counterstained with weak Mayer'shematoxylin solution for 2 min. Negative control slides were preparedwithout addition of primary antibody. The degree of expression ofchemokine was recorded independently by two expert observers as apercentage of cells positive for the chemokine based on a visualassessment of the intensity of brown reaction product within thecytoplasmic regions of each image on a scale of 0 (no staining) to3+(intense staining). To quantity the staining intensity, an averagescore was then calculated by summing the individual intensity levelscores.

In vivo image. To evaluate the stability of circulating IGNVs in mice,200 nmol of DiR dye-labeled IGNVs were injected into mice via the tailvein. Blood was drawn into an anti-coagulant tube at various time points(3 h, 24 h, 48 h, 72 h and 120 h) after the injection. The intensity ofDiR signals from equal volume blood samples were then measured using aKodak Image Station (4000 MM Pro system, Carestream, Woodbridge, Conn.)and quantified using the Carestream MI software.

To track the IGNVs in LPS-induced acute skin inflammation of mice, DiRdye labeled IGNVs (20, 40, 150, 300 nmol) were I.V. injected into mice.At 6 h and 24 h after the injection, images of living mice were obtainedusing a Kodak Image Station. Inflamed skin was then removed 24 h afterIGNV administration and the DiR dye signals in the skin were quantified.

To study targeted delivery of IGNVs in DSS-induced colitis mice, DiR dyelabeled IGNVs or GNVs (200 nmol) were I.V. injected into normal mice orcolitis mice having been provided 2.5% DSS in their drinking water for 5days, 24 h after a single I.V. injection, colons were collected andscanned using a Kodak Image Station and quantified using the vendorsoftware.

For biodistribution of IGNVs in tumor-bearing mice, mouse colon tumor(CT26) and breast tumor (4T1) bearing mice were I.V. administrated DiRdye labeled IGNVs (200 nmol) or GNVs (200 nmol). Whole body imaging wasdone at 6 h and 24 h after the injection. DiR dye signals in both CT26and 4T1 tumor tissues were also quantitatively measured 24 h after theinjection.

To determine the effects of chemokines on the homing of IGNVs, 200 nmolof DiR dye labeled IGNVs were pre-incubated with tissue extract or a 100ng of each of the recombinant chemokines, i.e., CXCL1, CXCL2, CXCL9,CXCL10, CCL2, CCL5, or combinations of CXCL1/2, CXCL9/10, CCL2/5 orCXCL1/2/9/10 plus CCL2/5 overnight at 4° C., IGNVs were then washed at100,000 g for 20 min at 4° C., resuspended in 100 μl PBS, and injectedinto LPS-induce acute skin inflammation mice or CT26 tumor-bearing mice,24 hr after the injection, skin or tumors were removed and the DiR dyesignals were quantitatively measured using a Kodak Image Station andquantified using the vendor software.

Cytokines assays. To evaluate the potential toxicity of IGNVs andcommercial liposomes, 200 nmol and 800 nmol of IGNVs or commercialliposomes as controls were I.V. injected into BALB/c mice daily for 3days. 24 h after the last injection, peripheral blood was collected andcytokines (TNF-α, IL-6 and IL-1β) were measured using ELISA kits.

To determine the effect of curcumin delivered by IGNVs on the inhibitionof induction of TNF-α, IL-6 and IL-1β, colon tissues from DSS-inducedcolitis mice were cultured ex vivo and TNF-α, IL-6 and IL-1β quantified.The colon tissues from DSS induced colitis mice that had been previouslyI.V. injected with PBS, GNVs (200 nmol), IGNVs (200 nmol), curcumin (50mg/kg), GNV-Cur (50 mg/kg) or IGNV-Cur (50 mg/kg) every 2 days for 5times were collected for ex vivo culture. In brief, the distal most 2 cmof the colon was washed with PBS containing penicillin/streptomycin andthen further cut into 1 cm² sections. Colon sections were cultured inserum free RPMI 1640 medium supplemented with penicillin/streptomycin.After 24 h in culture, cell-free supernatants were harvested and assayedfor cytokine secretion using ELISA kits (eBioscience).

To measure human chemokines released in cultured supernatants of SW620human colon cancer cells, 2×10⁵ cells/well were cultured in 6-wellplates. After 48 h in culture, the medium was collected and CCL2, CCL5and CXCL10 were analyzed using the ELISA MAX™ Deluxe Set from Biolegend.

Induction of local skin inflammation. Dermal inflammation of C57BL/6jmice (body weight=25 to 30 g) was induced by a single intradermalinjection in the flank region of 30 μg of LPS (Escherichia coli 0111:B4)in 50 μl of isotonic saline solution. 6 h after the injection, mice witha visible local skin inflammatory response were recruited for thisstudy.

DSS-induced colitis model. Colitis was induced using 2.5% (w/v) dextransodium sulfate that had been added to the drinking water. The DSSsolution was prepared fresh every other day. To assess the therapeuticeffects of curcumin delivered by IGNVs on colitis, beginning on day 3after mice were provided with 2.5% DSS in their drinking water, micewere I.V. injected with PBS, free GNVs (200 nmol), IGNVs (200 nmol),free curcumin (50 mg/kg), curcumin loaded GNVs (50 mg/kg) or curcuminloaded IGNVs (50 mg/kg) every 2 days for total of 5 injections. Bodyweight and the presence of blood in the stool were monitored daily.

Measurement of the concentration of curcumin in mice colon tissues.DSS-induced colitis mice were I.V. injected with PBS, GNVs, IGNVs, freecurcumin, GNV-Cur or IGNV-Cur every 2 days, the curcumin in colontissues was quantitatively analyzed using high performance liquidchromatography (HPLC) as previously described. In brief, colon tissueswere collected 6 h after last injection, weighed and placed in 1 ml PBSand homogenized. Fifty μl of citrate buffer solution was added to 0.8 mlof aliquots of the homogenized samples and vortexed for 30 s, followedby addition of 2 ml of ethyl acetate. The supematants were thencollected after 1000×g centrifugation for 5 min and dried under a streamof nitrogen gas. The obtained solid samples were re-dissolved in 100 μlof methanol and centrifuged at 10,000×g for 10 min. Twenty μl ofsupernatant were used for HPLC analysis. Curcumin was detected byreversed phase-HPLC with a DAD detector (Agilent 1100, USA) at a flowrate of 1 ml/min. The solvent system was (A) ddH₂O and (B) acetonitrilecontaining 0.1% TFA. The following gradients of mobile phase B were usedto run the column: 10-50% for 0-18 min, 50-90% for 18-25 min, 90% for25-30 min and 90-10% for 30-35 min.

CT26 and 4T1 tumor mice models. Xenograft tumor growth models were usedto demonstrate IGNV mediated targeted delivery of chemotherapy drug totumors versus free doxorubicin (Free DOX) or GNVDOX. In the first set ofexperiments, six-week-old female BALB/c mice were subcutaneouslyinjected with the murine colon cancer CT26 cell line (5.0×10⁵cells/mouse in 50 μl of PBS). In a second set of experiments,six-week-old female BALB/c mice were injected in a mammary fat pad withthe murine breast tumor 4T1 cell line (5.0×10⁵ cells/mouse in 50 μl ofPBS). When tumors reached approximately 60 mm³ in volume, the mice wererandomly assigned to different treatment groups and I.V. injected withfree GNVs, IGNVs, doxorubicin (DOX, 100 μg), GNVs loaded with DOX(GNV-DOX, 100 μg DOX ) or IGNVs loaded with DOX (IGNV-DOX, 100 μg DOX).Mice were treated every 3 days for 30 days. Growth of the tumors wasmeasured using a method as described previously.

Loading efficiency. To evaluate the loading efficiency of doxorubicinand curcumin in IGNVs, GNV-DOX or GNV-Cur particles were prepared byadding 200 μg of doxorubicin or curcumin into a grapefruit lipid film,and were subsequently sonicated using a method as described previously.To further make IGNV-DOX or IGNV-Cur, the GNV-DOX or GNV-Cur particlesformed through sonication as described above were mixed with EL4 cellplasma membrane-derived vesicles and then extruded 15-20 times throughan Avanti mini extruder with a 200 nm polycarbonate porous membrane. Theresulting suspension was centrifuged at 100,000×g for 30 min. Thesupernatants were collected and the residual doxorubicin, or curcumincontent was measured using a CARY 100 Bio UV-Visible Spectrophotometerat a wavelength of 497 and 426 nm, respectively. The loading efficiencywas calculated as follows: Loading efficiency=(Total drug-freedrug)/Total drug×100%.

Release profile of chemotherapy drugs. To test the in vitro profile ofdoxorubicin released from IGNV-DOX or DOX-NP™ purchased from AvantiPolar Lipids Inc. (Alabaster, Ala., USA), IGNV-DOX or DOX-NP™ with 200μg doxorubicin were suspended in 1 ml of PBS buffer solution atdiffering pH values of 5.0, 5.5, 6.0, 6.5 and 7.2. Each suspension wasplaced at 37° C. with shaking. At a predetermined time (0.5, 1, 2, 3, 6and 24 h), suspensions were centrifuged at 100,000×g for 30 min. Theamount of doxorubicin released from IGNVDOX or DOX-NP™ was measuredspectrophoiometrically at 497 nm. Release of curcumin from IGNV-Cur wasalso analyzed using a UV-Visible spectrophotometer set at 426 nm.

ATPlite assay. To test the potential cytotoxicity of IGNVs andcommercial DOX-NP™ control liposomes, 4T1 cells were cultured in 24-wellculture plates for 24 h with different doses (0, 10, 20, 40, 80, 160nmol) of IGNVs or control liposomes from Avanti Polar Lipids Inc.(Alabaster, Ala., USA). Cell cytotoxicity of IGNVs was measured usingthe Luminescence ATP Detection Assay System (PerkinElmer, MA, USA).Briefly, 200 μl of mammalian cell lysis buffer was added into each welland the plate shaken for 5 min. 50 μl cell lysis was dispensed into anOptiPlate, mixed with 50 μl of substrate solution, the plate shaken at700 rpm for 5 min, and the luminescence measured using a BioTek Synergy™HT Multidetection Microplate Reader with Gen 5 version 1.08 dataanalysis software (Winooski, Vt., USA).

Statistical analysis. One-way analysis of variance (ANOVA) followed byTurkey Post Hoc tests was used to determine the differences occurredbetween groups, and T test was used to determine the difference betweentwo groups (*p<0.05, **p<0.01 and ***p<0.001).

Example 1 Characterization of GNVs Coated with Inflammatory ChemokineReceptor Enriched Membrane Fraction of Activated T Cells (IGNVs).

IGNVs were generated by binding membranes derived from PMA activated EL4T cells to GNVs. IGNVs' morphology (FIG. 6A) and quantity of chemokinereceptors (FIG. 6B) from EL4 T cells stimulated with/without PMA on theIGNVs was analyzed. Vesicles from the purified middle band of a sucrosegradient (FIG. 7) were prepared by extrusion and subsequently bound withGNVs. The Zeta potential and size distribution of IGNVs was thenanalyzed (FIG. 1B). Transmission electron microscopy (TEM) imaging (FIG.1C) indicated that IGNVs shared a similar morphology with GNVs. IGNVswere internalized by CT26 colon cancer cells in a manner similar to GNVs(FIG. 1D), and co-localized with EL4 membrane (FIG. 1D, last column).Fluorescence resonance energy transfer (FRET) analysis further confirmedthat more than 83±2.2% of the GNVs were coated with plasma membrane fromactivated EL4 T cells (IGNVs) (FIG. 1E). After preparation and throughfive days at 22° C. (FIG. 9A) or 25 h at 37° C. (FIG. 9B) the IGNVs werestable without significant changes in size or charge.

Example 2 IGNVs Home to Inflammatory Sites

Next, to determine whether IGNVs would leave the peripheral circulationand home to inflammatory tissues, three inflammatory mouse models weretested. It was first sought to determine if IGNVs transmigrated througha HUVEC monolayer at a higher efficiency than GNVs by using an in vitrotranswell assay. The data demonstrated that much higher numbers of IGNVstransfected HUVECs (DAPI⁺PKH26⁺) migrated to the bottom of the transwell(right panel ) than GNVs (left panel) over a 48 h period (FIG. 2A).Addition of chemokines (CXCLI/2/9/10, CCL2/5) in the bottom of thetranswell further enhanced the efficiency of IGNV transmigration (FIG.2A, right panel, 4^(th) column, **p<0.01). Enhanced homing of IGNVs toinflammatory sites when compared with GNVs was further confirmed indifferent inflammatory models, two acute inflammation models (a LPSinduced skin inflammation (FIG. 2B), and DSS induced colitis (FIG. 2C,**p<0.01)) and two chronic inflammation cancer models (CT26 colon cancer(FIG. 2D, ***p<0.001) and 4T1 breast cancer (FIG. 2E, ***p<0.001)).

Example 3 Chemokine and Chemokine Receptors Play a Role in IGNV Homing

Leukocyte recruitment into inflamed tissue follows a well-definedcascade of events, beginning with capturing of free flowing leukocytesto the vessel wall, followed by rolling, adhesion to endothelial cells,post-adhesion strengthening, crawling, and finally transmigrationthrough endothelial junctions into sites of inflammation. During thesesteps, chemokines/chemokine receptors play a key role in the last step,transmigration. The profiles of chemokines from the extracts ofinflammatory tissues and the types of chemokine receptors coated on theIGNVs were therefore analyzed. Chemokine array data (FIG. 3A) indicatedthat chemokines identified are in much higher concentrations in theextracts of the inflammation models that were tested than the extractsfrom non-tumor mammary gland (FIG. 3A, normal). It was also noticed, ingeneral, that stronger chemokine signals were detected in the extractsfrom 4T1 breast tumor than from the skin of LPS induced skininflammation or CT26 colon tumor. FACS analysis data indicated thatcorresponding receptors of chemokines were detected on the IGNVs (FIG.3B). To determine which chemokine(s) in the inflammatory tissues play acausative role in recruiting IGNVs into the inflammatory tissue, an invitro transwell assay was conducted. The results indicated thetransmigration of IGNVs was remarkably affected by addition ofchemokinesinto the bottom of the transwells; whereas, there was no change in GNVsmigration with the addition ofchemokines. Addition of CXCL1 or CXCL2resulted in more IGNVs detected in the bottom of the well 48 hpost-addition, and the combination of chemokines, CXCL1/2/9/10,CCL2/5led to the highest efficiency in transmigration of IGNVs (FIG. 10). Theeffects of chemokines on IGNVs transmigration was then confirmed by theresults generated from a neutralizing chemokine assay. Althoughpre-incubation with recombinant chemokine against each chemokinereceptor partially attenuated the migration of IGNVs, neutralizing allsix chemokine receptors as listed or pre-incubation with 4T1 tumorextract led to a maximum reduction of IGNVs transmigration (FIG. 3C,*p<0.05, **p<0.01 and ***p<0.001). This reduction was also confirmed ina LPS induced skin inflammation model in mice where the mice were I.V.injected with DiR dye labeled IGNVs that had been pre-incubated with 4T1tumor extract (FIG. 3D, **p<0.01).

The data generated from a 24 h (time course was determined based on thedata from FIG. 3D) in vivo imaging analysis further confirmed thatalthough CXCL1, CCL2 and CCL5 have an effect, CXCL2 plays a key role inhoming IGNVs into inflammatory sites, including LPS induced acuteinflammation in skin (FIG. 3F, *p<0.05, **p<0.01 and ***p<0.001) and theCT26 colon cancer model (FIG. 3G, *p<0.05, **p<0.01 and ***p<0.001).

Integrins, including LFA-1 and α4β7, have multiple functions in theprocess of leukocyte recruitment from initially adhering to vessel wallsto crawling prior to the final step, transmigration. FACS data indicatedthat although no α4β7 was detected, LFA-1 was highly expressed on bothPMA-activated EL4 cells (FIG. 11) and present on IGNVs (FIG. 12).Transmigration of IGNVs was dramatically decreased in vitro (FIG. 3H,***p<0.001) and in vivo (FIG. 3I, *p<0.05) when LFA-1 on IGNVs wasneutralized. Since multiple factors including LFA-1 play a role in theprocess of leukocytes and nanoparticles homing to inflammatory sites,and chemokines/chemokine receptors has been known to play importantroles in the last step (transmigration) of leukocyte homing toinflammatory tissue in general, chemokine related assays were used as aprimary determinant of function without further analysis of the role ofIGNV LFA-1.

It was further determined whether the chemokines of interest wereup-regulated in human cancer tissues. The results fromimmunohistological staining of chemokines in human colon cancer(Table 1) and breast cancer (Table 2) suggested a much higher expressionof CXCL1, CXCL10, CCL2, and CCL5 in tumor tissues than in adjacentnon-tumor tissues (FIG. 3E, Tables 3,4).

TABLE 1 Characteristics of 20 Colon Cancer Patients. Characteristics nSex male 13 female 7 Age male 59.85 ± 3.027 female 56.88 ± 4.129Differentiation well/moderate 14 poor 6 Lymph node involvement yes 14 no6

TABLE 2 Characteristics of 21 Breast Cancer Patients. Characteristics nCases 21 Age 54.81 ± 2.126 Lymph node involvement yes  9 no 12

TABLE 3 IHC Scores of Chemokines (CXCL1, CXCL10, CCL2, and CCL5) inHuman Colon Cancer Tissues. Expression levels Chemokines − + ++ +++CXCL1 Adjacent 11 8 1 Tumor tissue 2 6 12 CXCL10 Adjacent 8 10 2 Tumortissue 1 8 11 CCL2 Adjacent 11 9 Tumor tissue 5 15 CCL5 Adjacent 11 9Tumor tissue 12 8

TABLE 4 IHC Scores of Chemokines (CXCL1, CXCL10, CCL2, and CCL5) inHuman Breast Cancer Tissues. Expression levels Chemokines − + ++ +++CXCL1 Adjacent 13 8 Tumor tissue 3 6 12 CXCL10 Adjacent 18 3 Tumortissue 2 10 9 CCL2 Adjacent 13 8 Tumor tissue 5 16 CCL5 Adjacent 18 3Tumor tissue 1 8 12

To further demonstrate whether the above-described approach could beapplied for treatment of patients in a personalized manner, GNVs coatedwith the membrane of LPS stimulated leukocytes isolated from theperipheral blood of healthy human subjects (FIG. 13A) or of mice (FIG.13B) were purified using a sucrose gradient. The chemokine receptors onthe IGNVs were quantitatively analyzed (FIGS. 4A-4B). It was thendetermined whether IGNVs were capable of homing to human tumor using thehuman colon SW620 tumor model since the chemokines of interest areoverexpressed in human colon cancer, as well as breast tumor tissue, andare also released from SW620 colon cancer cells (FIG. 14). The resultsfrom in vivo imaging analysis indicated that human SW620 tumor bearingmice (FIG. 4C, **p<0.01) or mice locally challenged with LPS (FIG. 4D,*p<0.05 and ***p<0.001) attracted more IGNVs than GNVs, and IGNVs coatedwith purified membranes of LPS stimulated leukocytes have the highestfluorescent intensity at day 5 after I.V. injection. The results from anin vitro transwell assay (FIG. 3C) prompted a further determination ofwhether CXCR2 plays a dominant role in IGNV homing to the inflammatorysite. The results generated from in vivo imaging analysis indicated thatIGNVs coated with the membrane of LPS stimulated leukocytes isolatedfrom the peripheral blood of CXCR2 knockout mice had significantlyattenuated the migration of IGNVs to the inflammatory site (FIG. 4E),suggesting that IGNV CXCR2 plays a key role in IGNV homing.

Example 4 In vivo Therapeutic Effects of Drugs Carried by IGNVs

Since no adverse side effects had been observed with an I.V. injectionof IGNVs (FIGS. 15A-15F), it was tested whether IGNVs can be used as atherapeutic drug delivery vehicle. IGNVs are capable of being loadedwith different drugs including chemo drugs, such as doxorubicin, andanti-inflammatory agents like curcumin (FIG. 16A). Both doxorubicin andcurcumin loaded IGNVs have a similar Zeta potential and sizedistribution (FIG. 16B, 16C) although the loading capacity (FIG. 16D)and releasing of an agent (FIG. 16E) is different for different types ofagents. Further analysis of the stability of IGNVs indicated thatcirculating IGNVs were stable and detectable until day 5 (FIG. 5A) afteran I.V. injection, which in turn provides a longer time span for IGNVsto home to where inflammation is occurring. In addition, IGNV-DOX werestable without releasing doxorubicin until they were in a pH of 5.5(FIG. 5B, **p<0.01) which is the pH in tumor tissue. In contrast, a pHas low as 5.0 did not result in the release of doxorubicin fromcommercially available doxorubicin loaded liposomes (FIG. 17A), althoughthere were no differences observed in the cell cytotoxicity (FIG. 17B),in vivo induction of pro-inflammatory cytokines (FIG. 17C), and therelease of liver enzymes (FIG. 17D) between IGNVs (FIG. 14) and controlliposomes. The tissue distribution of doxorubicin encapsulated intoIGNVs was determined next. The concentration of doxorubicin was higherin the tumor and lower in the liver of tumor bearing mice I.V. injectedwith IGNV-DOX when compared with DOX-NP™ treated mice (FIG. 5C,*p<0.05). This result was further confirmed by the much strongerintensity of doxorubicin signals detected in the CT26 tumor as well as4T1 breast tumor bearing mice I.V. injected with IGNV-DOX when comparedto mice treated with GNV-DOX or free doxorubicin (FIG. 5D). Injection ofGNV-DOX mixed with EL4 cell derived membrane vesicles had significantlylower levels of doxorubicin detected in the 4T1 tumor than IGNV-DOX(FIG. 18), suggesting that the extrusion of GNVs with the activatedleukocyte membranes, which leads to formation of IGNVs, is required forhigher efficiency delivery of DOX to inflammatory sites. The biologicaleffects of IGNV-DOX on the CT26 colon tumor and 4T1 breast tumor modelswere also significant when compared to the other treatments. IGNV-DOXtreatment led to significant inhibition in the growth of CT26 and 4T1tumor (FIG. 5E, *p<0.05, **p<0.01 and ***p<0.001) and extended thesurvival of tumor-bearing mice (FIG. 5F, *p<0.05, **p<0.01). The resultsfrom IGNV-Cur treatment of DSS induced mouse colitis also indicated thatIGNVs carrying curcumin has a better therapeutic effect on theinhibition of colitis than GNVs carrying curcumin or curcumin alone.This conclusion is supported by the fact that there was less blood instools (FIG. 19A), fewer leukocytes were observed infiltrating HEstained mouse colon tissue (FIG. 19B), there was less lost weight (FIG.19C) and there was a significantly improved survival rate (FIG. 19D) inDSS induced colitis mice I.V. injected with IGNV-Cur than control groupsas listed. These results were also consistent with higher concentrationof curcumin detected in the colon tissues of mice treated with IGNV-Cur(FIG. 19E). ELISA analysis further indicated that significantly lessTNF-α, IL-6 and IL-1β were detected in the colon tissue extracts of DSSinduced colitis mice I.V. injected with IGNV-Cur than with PBS/DSS, freeCur, or GNV-Cur (FIG. 20).

Discussion of Examples 1-4

The foregoing study describes an approach for targeted delivery oftherapeutic agents to inflammatory sites where the appropriate cells aretargeted; thereby promoting much more substantial therapeutic benefitswithout inducing adverse side-effects. It was shown that IGNVs can be aneffective, personalized approach to potentially treat patients with avariety of inflammatory conditions. The use of IGNVs avoids several ofthe problems that have arisen with conventional therapy vectors, such asthe lack of tissue targeting specificity, immunogenicity, difficulty inscalability and production, the need for life-long monitoring fortumorigenesis and other adverse clinical outcomes. Because IGNVs do notcause these concerns they have great potential as targeted deliveryvehicles, in particular, because production of GNVs is easily scaled upand the GNVs can be coated with leukocyte membranes from an individualmaking this approach personalized and economically feasible fortreatment of patients in low and middle income countries where they donot have comprehensive facilities to make synthetic therapeutic vectors.

To successfully target a specific tissue, four goals must at least bemet: 1) extended circulation of the delivery vector. 2) tissuepenetration by the vector, 3) tissue specificity and 4) release of thepayload, i.e., the therapeutic agent. Tumor tissues have abnormalmolecular and fluid transport dynamics, especially for macromoleculardrugs. This phenomenon is referred to as the “enhanced permeability andretention (EPR) effect” of macromolecules and lipids in solid tumors.Therefore, the longer the vector is in the circulation, the greateropportunity it has to penetrate the tumor tissue by utilizing the EPReffect. Previously published work shows that a nanovector made of lipidsfrom grapefruit nanoparticles circulates in the peripheral blood morethan 5 days in tumor bearing mice. GNVs are stable in the circulatorysystem and thus have a greater opportunity to enter the tumor tissue. Inaddition, the critical concentration of grapefruit nanoparticle derivedlipids for GNV formation was 1.5 μM (FIG. 21), and GNVs do not increasein size with increasing concentrations of total lipids up to 5 mM. Thediameter of GNVs at 25 mM-50 mM concentration and at 25° C. is increasedup to 200 nm, and retained at 200 nm while lipid concentration wasfurther increased up to 100 mM. It was believed that this finding notonly provides a guideline for selecting concentrations of grapefruitnanoparticle derived lipids to make desirable sized GNVs (50-200 nm) butalso a broad range of concentrations of grapefruit nanoparticle derivedlipids can be selected for making GNVs without leading to a furtherincrease of GNV size. The latter feature has an advantage in case that alarge amount of grapefruit nanoparticle derived lipids is required tomake GNVs which allows for higher concentrations of therapeutic agentsto be encapsulated per GNV. However, this feature of GNVs alone may notbe sufficient to deliver ample agent to the targeted tissue to have atherapeutic effect. Achieving specific and safe delivery of a drugacross endothelial cells is essential if it is to reach the targetedtissue. However, due to the fact that most delivery vectors lack anaffinity for endothelium, only a small fraction of therapeutics can bedelivered to the targeted tissue by utilizing the EPR effect. In theforegoing study, IGNVs were modified so they could take advantage of theactivated leukocyte pathway that is primarily driven bychemokines/chemokine receptors by coating the IGNVs with the membranesfrom activated leukocytes. This modification of IGNVs significantlyenhanced their endothelial cell transmigration capability so the IGNVscould enter inflammatory tissues. As was demonstrated in this study, notonly can this strategy be applied to breast cancer and colon cancer, butthe potential exists that the IGNVs could be used to treat manydifferent types of diseases since the inflammatory process is a hallmarkof many chronic diseases including cancer, infectious diseases, andautoimmune diseases. As shown in this study, the chemotherapy drugdoxorubicin and the anti-inflammatory agent curcumin can be deliveredsuccessfully by IGNVs to reach the desired inflammatory site and achievea therapeutic effect through modification of GNVs with membranes ofactivated leukocytes from individuals. Specifically, it was demonstratedthat I.V. injection of IGNV-DOX or IGNV-Cur significantly enhances theinhibition of breast tumor and colon tumor growth, and attenuates DSSinduced colitis, respectively. This response was due to improvedrecruitment of IGNVs into tumor as well as inflamed colon tissue. Themembranes from activated leukocytes were required to obtain thisbenefit, as it permitted IGNV delivery to the precise place whereinflammation is occurring.

It was also thought that other factors in addition tochemokines/chemokine receptors may play a role in IGNVs homing to aninflammatory site. Previous studies have suggested that LFA-1 plays arole in the nanoparticle homing to an inflamed site. The present studiesused LFA-1 (CD11a-CD18) as an example, and the foregoing data alsoshowed that CXCR2 and LFA-1 played a role in the transmigration of IGNVsas demonstrated in an in vitro trans-well blocking assay and in vivoskin inflammatory mouse model. Therefore, the role of IGNV chemokinereceptors as demonstrated in this study does not exclude a number ofother IGNV factors such as CXCR2 and LFA-1 which also plays a role inthe process of IGNV recruitment into inflamed tissue. This is also areason that it was believed IGNVs coated with total leukocyte membranesmay have a greater potential for being applied to personalized medicinefor targeted delivery to inflammatory sites than the use of individualchemokine receptor coated IGNVs. It was further conceivable thatmembrane associated chemokine and integrin profiles of circulatinginflammatory cells from patients with different chronic inflammatorydiseases may be different in their makeup. Furthermore, individualchemokine receptor coated GNVs may be potentially difficult to optimizein combinations or as a customized set or group of chemokine receptorsthat are most suitable for targeted delivery for an individual patient.Additionally there may be a higher cost for production of recombinantchemokine receptors and recombinant production would require FDAapproval for clinical use. Finally, potential biosafety issues couldarise due to using synthesized recombinant chemokine receptors.

A suitable delivery vehicle should be susceptible to manipulation sodelivered therapeutic drug can be released within targeted tissue.Evidence accumulated over the past decades has shown that the pH inelectrode-evaluated human tumor is on average lower than the pH ofnormal tissues. The results presented in the foregoing study show thatdoxorubicin encapsulated in the IGNVs is stable until the pH drops to6.0 or below. This feature of IGNVs allows the encapsulated drug toselectively be released in tumor tissue, and therefore reduces theside-effects seen when chemotherapy treatment non-discriminately affectshealthy organs and tissues, which is one of major obstacles forchemotherapy for treatment of cancer patients.

In the past decades, substantial experimental and clinical evidencesupports the conclusion that one of the most important mechanismsoperating in tumor progression involves chemokines and their receptors.Chemokines and their receptors not only play a role in cancer-relatedinflammation, but have been implicated in the invasiveness andmetastasis of diverse cancers. Without wishing to be bound by anyparticular theory, it was believed that pseudo-inflammatory chemokinereceptors delivered by GNVs may also act as soluble receptors to blockthe pathway(s) mediated by chemokine receptors expressed on the tumorcells or other tumor associated cells.

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It will be understood that various details of the presently disclosedsubject matter can be changed without departing from the scope of thesubject matter disclosed herein. Furthermore, the foregoing descriptionis for the purpose of illustration only, and not for the purpose oflimitation.

1. A composition, comprising: a microvesicle derived from an edible plant; and a plasma membrane coating the microvesicle, the plasma membrane derived from a targeting cell.
 2. The composition of claim 1, wherein the microvesicle encapsulates a therapeutic agent.
 3. The composition of claim 1, wherein the targeting cell is an activated leukocyte.
 4. The composition of claim 1, wherein the edible plant is a fruit or a vegetable.
 5. The composition of claim 1, wherein the fruit is selected from a grape, a grapefruit, and a tomato.
 6. The composition of claim 2, wherein the therapeutic agent is selected from a phytochemical agent and a chemotherapeutic agent.
 7. The composition of claim 6, wherein the therapeutic agent is a phytochemical agent, and wherein the phytochemical agent is selected from curcumin, resveratrol, baicalein, equol, fisetin, and quercetin.
 8. The composition of claim 6, wherein the therapeutic agent is a chemotherapeutic agent, and wherein the chemotherapeutic agent is selected from the group consisting of retinoic acid, 5-fluorouracil, vincristine, actinomycin D, adriamycin, cisplatin, docetaxel, doxorubicin, and taxol.
 9. The composition of claim 1, wherein the therapeutic agent comprises a nucleic acid molecule selected from an siRNA, a microRNA, and a mammalian expression vector.
 10. A pharmaceutical composition, comprising a composition according to claim 1 and a pharmaceutically-acceptable vehicle, carrier, or excipient.
 11. A method for treating an inflammatory disorder, comprising administering to a subject in need thereof an effective amount of a composition of claim
 1. 12. The method of claim 11, wherein the inflammatory disorder is selected from the group consisting of sepsis, septic shock, colitis, colon cancer, and arthritis.
 13. The method of claim 11, wherein the composition is administered orally or intransally.
 14. The method of claim 11, wherein administering the composition reduces an amount of an inflammatory cytokine in a subject.
 15. The method of claim 14, wherein the inflammatory cytokine is selected from the group consisting of tumor necrosis factor-α, interleukin-1β, interferon γ, and interleukin-6.
 16. The method of claim 11, wherein the edible plant is a fruit or a vegetable.
 17. The method of claim 16, wherein the fruit is selected from a grape, a grapefruit, and a tomato.
 18. The method of claim 11, wherein the microvesicle encapsulates a therapeutic agent, and wherein the therapeutic agent is a phytochemical agent.
 19. The method of claim 18, wherein the phytochemical agent is curcumin.
 20. The method of claim 11, wherein the inflammatory disorder is colitis.
 21. A method for treating a cancer in a subject, comprising administering to a subject an effective amount of a composition according to claim
 1. 22. The method of claim 21, wherein the microvesicle encapsulates a therapeutic agent, and wherein the therapeutic agent is selected from a phytochemical agent and a chemotherapeutic agent.
 23. The method of claim 21, wherein the cancer is selected from a brain cancer, a breast cancer, a lung cancer, and a colon cancer. 